Compositions and methods of treating amyotrophic lateral sclerosis (ALS)

ABSTRACT

The present invention relates to small interfering RNA (siRNA) molecules against the SOD1 gene, adeno-associated viral (AAV) vectors encoding siRNA molecules and methods for treating amyotrophic lateral sclerosis (ALS) using the siRNA molecules and AAV vectors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application which claims the benefit ofU.S. patent application Ser. No. 16/774,493, filed Jan. 28, 2020 andentitled Compositions and Methods of Treating Amyotrophic LateralSclerosis (ALS); which is a divisional application which claims thebenefit of U.S. patent application Ser. No. 15/526,690, filed May 12,2017 and entitled Compositions and Methods of Treating AmyotrophicLateral Sclerosis (ALS); which is a national stage filing under 35U.S.C. § 371 of International Application No. PCT/US2015/060562, filedNov. 13, 2015 and entitled Compositions and Methods of TreatingAmyotrophic Lateral Sclerosis (ALS); which claims priority to U.S.Provisional Patent Application No. 62/079,588, entitled Treatment ofAmyotrophic Lateral Sclerosis (ALS) with siRNAs targeting SOD-1, filedNov. 14, 2014, U.S. Provisional Patent Application No. 62/211,992,entitled Compositions and Methods of Treating Amyotrophic LateralSclerosis (ALS), filed Aug. 31, 2015, U.S. Provisional PatentApplication No. 62/234,466, entitled Compositions and Methods ofTreating Amyotrophic Lateral Sclerosis (ALS), filed Sep. 29, 2015; thecontents of each of which are herein incorporated by reference in theirentirety.

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled20571011USDIV2_SL.txt, created on Jan. 6, 2021, which is 126, 990 bytesin size. The information in the electronic format of the sequencelisting is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to compositions, methods and processes forthe design, preparation, manufacture, use and/or formulation ofmodulatory polynucleotides, e.g., small interfering RNA (siRNA)molecules which target the superoxide dismutase 1 (SOD1) gene. As usedherein, a “modulatory polynucleotide” is any nucleic acid sequence(s)which functions to modulate (either increase or decrease) the level oramount of a target gene, e.g., mRNA or protein levels. Targeting of theSOD1 gene may interfere with SOD1 gene expression and SOD1 enzymeproduction. In some embodiments, the nucleic acid sequence encoding thesiRNA molecule are inserted into recombinant adeno-associated virus(AAV) vectors. Methods for using the siRNA molecules to inhibit SOD1gene expression in a subject with a neurodegenerative disease (e.g.,amyotrophic lateral sclerosis (ALS)) are also disclosed.

BACKGROUND OF THE INVENTION

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease,is the most fatal progressive neurodegenerative disease, characterizedby the predominant loss of motor neurons (MNs) in primary motor cortex,the brainstem, and the spinal cord. The loss of motor neurons devastatesbasic, fundamental movements, such as breathing, and typically causesdeath to patients within 2-5 years after diagnosis. Progressivedeterioration of motor function in patients severely disrupts theirbreathing ability, requiring some form of breathing aid for survival ofthe patients. Other symptoms also include muscle weakness in hands,arms, legs or the muscles of swallowing. Some patients (e.g., FTD-ALS)may also develop frontotemporal dementia.

According to the ALS Association, approximately 5,600 people in theUnited States of America are diagnosed with ALS each year. The incidenceof ALS is two per 100,000 people, and it is estimated that as many as30,000 Americans may have the disease at any given time.

Two forms of ALS have been described: one is sporadic ALS (sALS), whichis the most common form of ALS in the United States of America andaccounts for 90 to 95% of all cases diagnosed; the other is familial ALS(fALS), which occurs in a family lineage mainly with a dominantinheritance and only accounts for about 5 to 10% of all cases in theUnited States of America. sALS and fALS are clinicallyindistinguishable.

Pathological studies found that disturbance of some cellular processesoccur after disease onset, including increased ER stress, generation offree radicals (i.e., reactive oxygen species (ROS)), mitochondrialdysfunction, protein aggregation, apoptosis, inflammation, and glutamateexcitotoxicity, specifically in the motor neurons (MNs).

The causes of ALS are complicated and heterogeneous. In general, ALS isconsidered to be a complex genetic disorder in which multiple genes incombination with environmental exposures combine to render a personsusceptible. More than a dozen genes associated with ALS have beendiscovered, including, SOD-1 (Cu²⁺/Zn²⁺ superoxide dismutase), TDP-43(TARDBP, TAR DNA binding protein-43), FUS (Fused in Sarcoma/Translocatedin Sarcoma), ANG (Angiogenin), ATXN2 (Ataxin-2), valosin containingprotein (VCP), OPTN (Optineurin) and an expansion of the noncodingGGGGCC hexanucleotide repeat in the chromosome 9, open reading frame 72(C9ORF72). However, the exact mechanisms of motor neuron degenerationare still elusive.

Currently, there is no curative treatment for ALS. The only FDA approveddrug is Riluzole, which antagonizes the glutamate response to reduce thepathological development of ALS. However, only about a three-month lifespan expansion for ALS patients in the early stages has been reported,and no therapeutic benefit for ALS patients in the late stages has beenobserved, indicating a lack of therapeutic options for the patients(Bensimon G et al., J Neurol. 2002, 249, 609-615). Therefore, a newtreatment strategy that can effectively prevent the disease progressionis still in demand.

Many different strategies are under investigation for potentialtreatment of both sporadic and familial ALS. One strategy is based onthe neuroprotective and/or regenerative effect of neurotrophic factors,such as Insulin-like growth factor I (IGF-I), Glial cell line-derivedneurotrophic factor (GDNF), Vascular endothelial growth factor (VEGF),Colivelin and Activity dependent neurotrophic factor (ADNF) derivedpeptide, which can promote neuronal survival. Several studiesdemonstrated that neurotrophic factors can preserve motor neuronfunctionality, therefore improving motor performance in the SOD1transgenic mice. However, such treatment often fails to prolong thesurvival of SOD1 mice, suggesting that neurotrophic factors are notsufficient to prolong neuronal survival (See a review by Yacila andSari, Curr Med Chem., 2014, 21(31), 3583-3593).

Another strategy for ALS treatment has focused on stem cell-basedtherapy. Stem cells have the potential to generate motor neurons,thereby replacing degenerating motor neurons in the ALS-affected CNSsuch as primary motor cortex, brainstem, and spinal cord. Stem cellsderived from multiple sources have been investigated, including inducedpluripotent stem cells (iPSCs), mesenchymal stromal cells (MSCs) (e.g.,bone marrow mesenchymal stromal cells (BMSCs) and adipocyte stem cells(ASCs)) and neural tissue origin neural stem cells (e.g., fetal spinalneural stem cells (NSCs), multipotent neural progenitor cells (NPCs))(e.g., reviewed by Kim C et al., Exp. Neurobiol., 2014, 23(3), 207-214).

Mutations in the gene of superoxide dismutase type I (SOD1; Cu²⁺/Zn²⁺superoxide dismutase type I) are the most common cause of fALS,accounting for about 20 to 30% of all fALS cases. Recent reportsindicate that SOD1 mutations may also be linked to about 4% of all sALScases (Robberecht and Philip, Nat. Rev. Neurosci., 2013, 14, 248-264).SOD1-linked fALS is most likely not caused by loss of the normal SOD1activity, but rather by a gain of a toxic function. One of thehypotheses for mutant SOD1-linked fALS toxicity proposes that anaberrant SOD1 enzyme causes small molecules such as peroxynitrite orhydrogen peroxide to produce damaging free radicals. Other hypothesesfor mutant SOD1 neurotoxicity include inhibition of the proteasomeactivity, mitochondrial damage, disruption of RNA processing andformation of intracellular aggregates. Abnormal accumulation of mutantSOD1 variants and/or wild-type SOD1 in ALS forms insoluble fibrillaraggregates which are identified as pathological inclusions. AggregatedSOD1 protein can induce mitochondria stress (Vehvilainen P et al., FrontCell Neurosci., 2014, 8, 126) and other toxicity to cells, particularlyto motor neurons.

These findings indicate that SOD1 can be a potential therapeutic targetfor both familial and sporadic ALS. A therapy that can reduce the SOD1protein produced in the central nervous system of ALS patients mayameliorate the symptoms of ALS in patients such as motor neurondegeneration and muscle weakness and atrophy. Agents and methods thataim to prevent the formation of wild type and/or mutant SOD1 proteinaggregation may prevent disease progression and allow for ameliorationof ALS symptoms. RNA interfering (RNAi) mediated gene silencing hasdrawn researchers' interest in recent years. Small double stranded RNA(small interfering RNA) molecules that target the SOD1 gene haven beentaught in the art for their potential in treating ALS (See, e.g., U.S.Pat. No. 7,632,938 and U.S. Patent Publication No. 20060229268, thecontents of which is herein incorporated by reference in its entirety).

The present invention develops an RNA interference-based approach toinhibit or prevent the expression of SOD1 in ALS patients for treatmentof the disease.

The present invention provides novel double stranded RNA (dsRNA)constructs and siRNA constructs and methods of their design. Inaddition, these novel siRNA constructs may be synthetic molecules or beencoded in an expression vector (one or both strands) for delivery intocells. Such vectors include, but are not limited to adeno-associatedviral vectors such as vector genomes of any of the AAV serotypes orother viral delivery vehicles such as lentivirus, etc.

SUMMARY OF THE INVENTION

The present invention relates to RNA molecule mediated gene specificinterference with gene expression and protein production. Methods fortreating motor neuron degeneration diseases such as amyotrophic lateralsclerosis are also included in the present invention. The siRNA includedin the compositions featured herein encompass a dsRNA having anantisense strand (the antisense strand) having a region that is 30nucleotides or less, generally 19-24 nucleotides in length, that issubstantially complementary to at least part of an mRNA transcript ofthe SOD1 gene.

The present invention provides short double stranded RNA molecules suchas small interfering RNA (siRNA) duplexes that target SOD1 mRNA tointerfere with SOD1 gene expression and/or SOD1 protein production. ThesiRNA duplexes of the present invention may interfere with both allelesof the SOD1 gene irrespective of any particular mutation in the SOD1gene, and may particularly interact with those found in ALS disease.

In some embodiments, such siRNA molecules, or a single strand of thesiRNA molecules, are inserted into adeno-associated viral vectors to beintroduced into cells, specifically motor neurons and/or othersurrounding cells in the central nervous system.

The siRNA duplex of the present invention comprises an antisense strandand a sense strand hybridized together forming a duplex structure,wherein the antisense strand is complementary to the nucleic acidsequence of the targeted SOD1 gene, and wherein the sense strand ishomologous to the nucleic acid sequence of the targeted SOD1 gene. Insome aspects, the 5′ end of the antisense strand has a 5′ phosphategroup and the 3′ end of the sense strand contains a 3′ hydroxyl group.In other aspects, there are none, one or 2 nucleotides overhangs at the3′ end of each strand.

According to the present invention, each strand of the siRNA duplextargeting the SOD1 gene is about 19-25 nucleotides in length, preferablyabout 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23nucleotides, 24 nucleotides, or 25 nucleotides in length. In someaspects, the siRNAs may be unmodified RNA molecules.

In other aspects, the siRNAs may contain at least one modifiednucleotide, such as base, sugar or backbone modification.

In one embodiment, an siRNA or dsRNA includes at least two sequencesthat are complementary to each other. The dsRNA includes a sense strandhaving a first sequence and an antisense strand having a secondsequence. The antisense strand includes a nucleotide sequence that issubstantially complementary to at least part of an mRNA encoding SOD1,and the region of complementarity is 30 nucleotides or less, and atleast 15 nucleotides in length. Generally, the dsRNA is 19 to 24, e.g.,19 to 21 nucleotides in length. In some embodiments the dsRNA is fromabout 15 to about 25 nucleotides in length, and in other embodiments thedsRNA is from about 25 to about 30 nucleotides in length.

The dsRNA, either upon contacting with a cell expressing SOD1 or upontranscription within a cell expressing SOD1, inhibits or suppresses theexpression of a SOD1 gene by at least 10%, at least 20%, at least 25%,at least 30%, at least 35% or at least 40% or more, such as when assayedby a method as described herein.

According to the present invention, AAV vectors comprising the nucleicacids encoding the siRNA duplexes, one strand of the siRNA duplex or thedsRNA targeting SOD1 gene are produced, the AAV vector serotype may beAAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47,AAV9(hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ8 and/or AAV-DJ,and variants thereof.

According to the present invention, siRNA duplexes or dsRNA targetingthe SOD1 gene in ALS are selected from the siRNA duplexes listed inTable 3, 11 or 13. Preferably, the siRNA duplexes or dsRNA targetingSOD1 gene in ALS are selected from the group consisting of siRNAduplexes: D-2757, D-2806, D-2860, D-2861, D-2875, D-2871, D-2758,D-2759, D-2866, D-2870, D-2823 and D-2858.

The present invention also provides pharmaceutical compositionscomprising at least one siRNA duplex targeting the SOD1 gene and apharmaceutically acceptable carrier. In some aspects, a nucleic acidsequence encoding the siRNA duplex is inserted into an AAV vector.

In some embodiments, the present invention provides methods forinhibiting/silencing SOD1 gene expression in a cell. Accordingly, thesiRNA duplexes or dsRNA can be used to substantially inhibit SOD1 geneexpression in a cell, in particular in a motor neuron. In some aspects,the inhibition of SOD1 gene expression refers to an inhibition by atleast about 20%, preferably by at least about 30%, 40%, 50%, 60%, 70%,80%, 85%, 90%, 95% and 100%. Accordingly, the protein product of thetargeted gene may be inhibited by at least about 20%, preferably by atleast about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. TheSOD1 gene can be either a wild type gene or a mutated SOD1 gene with atleast one mutation. Accordingly, the SOD1 protein is either wild typeprotein or a mutated polypeptide with at least one mutation.

In some embodiments, the present invention provides methods fortreating, or ameliorating amyotrophic lateral sclerosis associated withabnormal SOD1 gene and/or SOD1 protein in a subject in need oftreatment, the method comprising administering to the subject apharmaceutically effective amount of at least one siRNA duplex targetingthe SOD1 gene, delivering said siRNA duplex into targeted cells,inhibiting SOD1 gene expression and protein production, and amelioratingsymptoms of ALS in the subject.

In some embodiments, an AAV vector comprising the nucleic acid sequenceencoding at least one siRNA duplex targeting the SOD1 gene isadministered to the subject in need for treating and/or amelioratingALS. The AAV vector serotype may be selected from the group consistingof AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47,AAV9(hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrh10 and AAV-DJ, andvariants thereof.

In some aspects, ALS is familial ALS linked to SOD1 mutations. In otheraspects, ALS is sporadic ALS which is characterized by abnormalaggregation of SOD1 protein or disruption of SOD1 protein function orlocalization, though not necessarily as a result of genetic mutation.The symptoms of ALS ameliorated by the present method may include motorneuron degeneration, muscle weakness, stiffness of muscles, slurredspeech and/or difficulty in breathing.

In some embodiments, the siRNA duplexes or dsRNA targeting SOD1 gene orthe AAV vectors comprising such siRNA-encoding molecules may beintroduced directly into the central nervous system of the subject, forexample, by intracranial injection.

In some embodiments, the pharmaceutical composition of the presentinvention is used as a solo therapy. In other embodiments, thepharmaceutical composition of the present invention is used incombination therapy. The combination therapy may be in combination withone or more neuroprotective agents such as small molecule compounds,growth factors and hormones which have been tested for theirneuroprotective effect on motor neuron degeneration.

In some embodiments, the present invention provides methods fortreating, or ameliorating amyotrophic lateral sclerosis by administeringto a subject in need thereof a therapeutically effective amount of aplasmid or AAV vector described herein. The ALS may be familial ALS orsporadic ALS.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages will beapparent from the following description of particular embodiments of theinvention, as illustrated in the accompanying drawings in which likereference characters refer to the same parts throughout the differentviews. The drawings are not necessarily to scale, emphasis instead beingplaced upon illustrating the principles of various embodiments of theinvention.

FIG. 1 is a histogram showing the activity of the constructs encoded inan AAV vector.

FIG. 2 is a histogram showing the activity of the guide strand of themodulatory polynucleotides encoded in an AAV vector in HEK293T cells.

FIG. 3 is a histogram showing the activity of the passenger strand ofthe modulatory polynucleotides encoded in an AAV vector in HEK293Tcells.

FIG. 4 is a histogram showing the activity of the guide strand of themodulatory polynucleotides encoded in an AAV vector in HeLa cells.

FIG. 5 is a histogram showing the activity of the passenger strand ofthe modulatory polynucleotides encoded in an AAV vector in HeLa cells.

FIG. 6 is a histogram for the intracellular AAV DNA.

FIG. 7 is a histogram showing the activity of the constructs encoded inan AAV vector in human motor neurons.

FIG. 8 is a chart showing the dose-dependent silencing of SOD1 in U251MGcells.

FIG. 9 is a chart showing the dose-dependent silencing of SOD1 in humanastrocyte cells.

FIG. 10 is a chart showing the time course of the silencing of SOD1 inU251MG cells.

FIG. 11A, FIG. 11B and FIG. 11C are charts showing the dose-dependenteffects of a construct. FIG. 11A shows the relative SOD1 expression.FIG. 11B shows the percent of guide strand. FIG. 11C shows the percentof the passenger strand.

FIG. 12 is a diagram showing the location of the modulatorypolynucleotide (MP) in relation to the ITRs, the intron (I) and thepolyA (P).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to modulatory polynucleotides, e.g., RNAor DNA molecules as therapeutic agents. RNA interfering mediated genesilencing can specifically inhibit targeted gene expression. The presentinvention then provides small double stranded RNA (dsRNA) molecules(small interfering RNA, siRNA) targeting the SOD1 gene, pharmaceuticalcompositions comprising such siRNAs, as well as processes of theirdesign. The present invention also provides methods of their use forinhibiting SOD1 gene expression and protein production, for treatingneurodegenerative disease, in particular, amyotrophic lateral sclerosis(ALS).

The present invention provides small interfering RNA (siRNA) duplexes(and modulatory polynucleotides encoding them) that target SOD1 mRNA tointerfere with SOD1 gene expression and/or SOD1 protein production. ThesiRNA duplexes of the present invention may interfere with both allelesof the SOD1 gene irrespective of any particular mutation in the SOD1gene, and may particularly interact with those found in ALS disease.

In some embodiments, a nucleic acid sequence encoding such siRNAmolecules, or a single strand of the siRNA molecules, is inserted intoadeno-associated viral vectors and introduced into cells, specificallymotor neurons and/or other surrounding cells in the central nervoussystem.

The encoded siRNA duplex of the present invention contains an antisensestrand and a sense strand hybridized together forming a duplexstructure, wherein the antisense strand is complementary to the nucleicacid sequence of the targeted SOD1 gene, and wherein the sense strand ishomologous to the nucleic acid sequence of the targeted SOD1 gene. Insome aspects, the 5′ end of the antisense strand has a 5′ phosphategroup and the 3′ end of the sense strand contains a 3′ hydroxyl group.In other aspects, there are none, one or 2 nucleotide overhangs at the3′ end of each strand.

According to the present invention, each strand of the siRNA duplextargeting the SOD1 gene is about 19 to 25, 19 to 24 or 19 to 21nucleotides in length, preferably about 19 nucleotides, 20 nucleotides,21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25nucleotides in length. In some aspects, the siRNAs may be unmodified RNAmolecules.

In other aspects, the siRNAs may contain at least one modifiednucleotide, such as base, sugar or backbone modification.

In one embodiment, an siRNA or dsRNA includes at least two sequencesthat are complementary to each other. The dsRNA includes a sense strandhaving a first sequence and an antisense strand having a secondsequence. The antisense strand includes a nucleotide sequence that issubstantially complementary to at least part of an mRNA encoding SOD1,and the region of complementarity is 30 nucleotides or less, and atleast 15 nucleotides in length. Generally, the dsRNA is 19 to 25, 19 to24 or 19 to 21 nucleotides in length. In some embodiments the dsRNA isfrom about 15 to about 25 nucleotides in length, and in otherembodiments the dsRNA is from about 25 to about 30 nucleotides inlength.

The dsRNA, whether directly administered or encoded in an expressionvector upon contacting with a cell expressing SOD1, inhibits theexpression of SOD1 by at least 10%, at least 20%, at least 25%, at least30%, at least 35% or at least 40% or more, such as when assayed by amethod as described herein.

The siRNA molecules included in the compositions featured hereincomprise a dsRNA having an antisense strand (the antisense strand)having a region that is 30 nucleotides or less, generally 19 to 25, 19to 24 or 19 to 21 nucleotides in length, that is substantiallycomplementary to at least part of an mRNA transcript of the SOD1 gene.

According to the present invention, AAV vectors comprising the nucleicacids of the siRNA duplexes, one strand of the siRNA duplex or the dsRNAtargeting SOD1 gene are produced, the AAV vector serotypes may be AAV1,AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14),AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ8 and AAV-DJ, and variantsthereof.

According to the present invention, siRNA duplexes or the encoded dsRNAtargeting the SOD1 gene in ALS is selected from the siRNA duplexeslisted in Table 3. In some embodiments, the siRNA duplexes or dsRNAtargeting the SOD1 gene in ALS is selected from the group consisting ofsiRNA duplexes: D-2757, D-2806, D-2860, D-2861, D-2875, D-2871, D-2758,D-2759, D-2866, D-2870, D-2823 and D-2858.

The present invention also provides pharmaceutical compositionscomprising at least one siRNA duplex targeting the SOD1 gene and apharmaceutically acceptable carrier. In some aspects, the siRNA duplexis encoded by an AAV vector.

In some embodiments, the present invention provides methods forinhibiting/silencing SOD1 gene expression in a cell. Accordingly, thesiRNA duplexes or encoded dsRNA can be used to substantially inhibitSOD1 gene expression in a cell, in particular in a motor neuron. In someaspects, the inhibition of SOD1 gene expression refers to an inhibitionby at least about 20%, such as by at least about 30%, 40%, 50%, 60%,70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%,20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%,30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%,40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%,50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%,70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100%.Accordingly, the protein product of the targeted gene may be inhibitedby at least about 20%, preferably by at least about 30%, 40%, 50%, 60%,70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%,20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%,30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%,40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%,50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%,70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100%.The SOD1 gene can be either a wild type gene or a mutated SOD1 gene withat least one mutation. Accordingly, the SOD1 protein is either wild typeprotein or a mutated polypeptide with at least one mutation.

In one embodiment, the siRNA duplexes or encoded dsRNA may be used toreduce the expression of SOD1 protein by at least about 30%, 40%, 50%,60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%,20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%,30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%,40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%,50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%,70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100%. Asa non-limiting example, the expression of SOD1 protein expression may bereduced 50-90%.

In one embodiment, the siRNA duplexes or encoded dsRNA may be used toreduce the expression of SOD1 mRNA by at least about 30%, 40%, 50%, 60%,70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%,20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%,30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%,40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%,50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%,70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100%. Asa non-limiting example, the expression of SOD1 mRNA expression may bereduced 50-90%.

In one embodiment, the siRNA duplexes or encoded dsRNA may be used toreduce the expression of SOD1 protein and/or mRNA in at least one regionof the CNS such as, but not limited to the spinal cord, the forebrain,the midbrain or the hindbrain. The expression of SOD1 protein and/ormRNA is reduced by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%,90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%,20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%,30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%,40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%,60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%,80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100% in at least oneregion of the CNS. As a non-limiting example, the expression of SOD1protein and mRNA in the spinal cord is reduced by 50-90%.

In some embodiments, the present invention provides methods fortreating, or ameliorating amyotrophic lateral sclerosis associated withabnormal SOD1 gene and/or SOD1 protein in a subject in need oftreatment, the method comprising administering to the subject apharmaceutically effective amount of at least one siRNA duplex or anucleic acid encoding an siRNA duplex targeting the SOD1 gene,delivering said siRNA duplex (or encoded duplex) into targeted cells,inhibiting SOD1 gene expression and protein production, and amelioratingsymptoms of ALS in the subject.

In some embodiments, an AAV vector comprising the nucleic acid sequenceof at least one siRNA duplex targeting the SOD1 gene is administered tothe subject in need for treating and/or ameliorating ALS. The AAV vectorserotype may be selected from the group consisting of AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11,AAV12, AAVrh8, AAVrh10, AAV-DJ8 (AAVDJ8) and AAV-DJ (AAVDJ), andvariants thereof. In one embodiment, the AAV vector serotype is AAV2. Inanother embodiment, the AAV vector is AAVDJ. In yet another embodiment,the AAV vector serotype is AAVDJ8.

In one embodiment, the serotype which may be useful in the presentinvention may be AAV-DJ8. The amino acid sequence of AAV-DJ8 maycomprise two or more mutations in order to remove the heparin bindingdomain (HBD). As a non-limiting example, the AAV-DJ sequence describedas SEQ ID NO: 1 in U.S. Pat. No. 7,588,772, the contents of which areherein incorporated by reference in their entirety, may comprise twomutations: (1) R587Q where arginine (R; arg) at amino acid 587 ischanged to glutamine (Q; Gln) and (2) R590T where arginine (R; Arg) atamino acid 590 is changed to threonine (T; Thr). As another non-limitingexample, may comprise three mutations: (1) K406R where lysine (K; Lys)at amino acid 406 is changed to arginine (R; Arg), (2) R587Q wherearginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gln) and(3) R590T where arginine (R; Arg) at amino acid 590 is changed tothreonine (T; Thr).

In some aspects, ALS is familial ALS linked to SOD1 mutations. In otheraspects, ALS is sporadic ALS which is characterized by abnormalaggregation of SOD1 protein or aberrations in SOD1 protein function andlocalization. The symptoms of ALS ameliorated by the present method mayinclude, but are not limited to, motor neuron degeneration, muscleweakness, stiffness of muscles, slurred speech and/or difficulty inbreathing.

In some embodiments, the siRNA duplexes or encoded dsRNA targeting theSOD1 gene or the AAV vectors comprising such siRNA molecules may beintroduced directly into the central nervous system of the subject, forexample, by intracranial injection.

In some embodiments, the pharmaceutical composition of the presentinvention is used as a solo therapy. In other embodiments, thepharmaceutical composition of the present invention is used incombination therapy. The combination therapy may be in combination withone or more neuroprotective agents such as small molecule compounds,growth factors and hormones which have been tested for theirneuroprotective effect on motor neuron degeneration.

In some embodiments, the present invention provides methods fortreating, or ameliorating amyotrophic lateral sclerosis by administeringto a subject in need thereof a therapeutically effective amount of aplasmid or AAV vector described herein. The ALS may be familial ALS orsporadic ALS.

The details of one or more embodiments of the invention are set forth inthe accompanying description below. Although any materials and methodssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred materialsand methods are now described. Other features, objects and advantages ofthe invention will be apparent from the description. In the description,the singular forms also include the plural unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. In the case of conflict, the present description will control.

Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS), an adult-onset neurodegenerativedisorder, is a progressive and fatal disease characterized by theselective death of motor neurons in the motor cortex, brainstem andspinal cord. The incidence of ALS is about 1.9 per 100,000. Patientsdiagnosed with ALS develop a progressive muscle phenotype characterizedby spasticity, hyperreflexia or hyporeflexia, fasciculations, muscleatrophy and paralysis. These motor impairments are caused by thedenervation of muscles due to the loss of motor neurons. The majorpathological features of ALS include degeneration of the corticospinaltracts and extensive loss of lower motor neurons (LMNs) or anterior horncells (Ghatak et al., J Neuropathol Exp Neurol., 1986, 45, 385-395),degeneration and loss of Betz cells and other pyramidal cells in theprimary motor cortex (Udaka et al., Acta Neuropathol, 1986, 70, 289-295;Maekawa et al., Brain, 2004, 127, 1237-1251) and reactive gliosis in themotor cortex and spinal cord (Kawamata et al., Am J Pathol., 1992, 140,691-707; and Schiffer et al., J Neurol Sci., 1996, 139, 27-33). ALS isusually fatal within 3 to 5 years after the diagnosis due to respiratorydefects and/or inflammation (Rowland L P and Shneibder N A, N Engl. JMed., 2001, 344, 1688-1700).

A cellular hallmark of ALS is the presence of proteinaceous,ubiquitinated, cytoplasmic inclusions in degenerating motor neurons andsurrounding cells (e.g., astrocytes). Ubiquitinated inclusions (i.e.,Lewy body-like inclusions or Skein-like inclusions) are the most commonand specific type of inclusion in ALS and are found in LMNs of thespinal cord and brainstem, and in corticospinal upper motor neurons(UMNs) (Matsumoto et al., J Neurol Sci., 1993, 115, 208-213; and Sasakand Maruyama, Acta Neuropathol., 1994, 87, 578-585). A few proteins havebeen identified to be components of the inclusions, including ubiquitin,Cu/Zn superoxide dismutase 1 (SOD1), peripherin and Dorfin.Neurofilamentous inclusions are often found in hyaline conglomerateinclusions (HCIs) and axonal ‘spheroids’ in spinal cord motor neurons inALS. Other types and less specific inclusions include Bunina bodies(cystatin C-containing inclusions) and Crescent shaped inclusions (SCIs)in upper layers of the cortex. Other neuropathological features seen inALS include fragmentation of the Golgi apparatus, mitochondrialvacuolization, and ultrastructural abnormalities of synaptic terminals(Fujita et al., Acta Neuropathol. 2002, 103, 243-247).

In addition, in frontotemporal dementia ALS (FTD-ALS) cortical atrophy(including the frontal and temporal lobes) is also observed, which maycause cognitive impairment in FTD-ALS patients.

ALS is a complex and multifactorial disease and multiple mechanismshypothesized as responsible for ALS pathogenesis include, but are notlimited to, dysfunction of protein degradation, glutamateexcitotoxicity, mitochondrial dysfunction, apoptosis, oxidative stress,inflammation, protein misfolding and aggregation, aberrant RNAmetabolism, and altered gene expression.

About 10%-15% of ALS cases have family history of the disease, and thesepatients are referred to as familial ALS (fALS) or inherited patients,commonly with a Mendelian dominant mode of inheritance and highpenetrance. The remainder (approximately 85%-95%) is classified assporadic ALS (sALS), as they are not associated with a documented familyhistory, but instead are thought to be due to other risk factorsincluding, but not limited to environmental factors, geneticpolymorphisms, somatic mutations, and possibly gene-environmentalinteractions. In most cases, familial (or inherited) ALS is inherited asautosomal dominant disease, but pedigrees with autosomal recessive andX-linked inheritance and incomplete penetrance exist. Sporadic andfamilial forms are clinically indistinguishable suggesting a commonpathogenesis. The precise cause of the selective death of motor neuronsin ALS remains elusive. Progress in understanding the genetic factors infALS may shed light on both forms of the disease.

Recently, an explosion to genetic causes of ALS has discovered mutationsin more than 10 different genes that are known to cause fALS. The mostcommon ones are found in the genes encoding Cu/Zn superoxide dismutase 1(SOD1; ˜20%) (Rosen D R et al., Nature, 1993, 362, 59-62), fused insarcoma/translated in liposarcoma (FUS/TLS; 1-5%) and TDP-43 (TARDBP;1-5%). Recently, a hexanucleotide repeat expansion (GGGGCC)_(n) in theC9orF72 gene was identified as the most frequent cause of fALS (˜40%) inthe Western population (reviewed by Renton et al., Nat. Neurosci., 2014,17, 17-23). Other genes mutated in ALS include alsin (ALS2), senataxin(SETX), vesicle-associated membrane protein (VAPB), and angiogenin(ANG). fALS genes control different cellular mechanisms, suggesting thatthe pathogenesis of ALS is complicated and may be related to severaldifferent processes finally leading to motor neuron degeneration.

SOD1 is one of the three human superoxide dismutases identified andcharacterized in mammals: copper-zinc superoxide dismutase (Cu/ZnSOD orSOD1), manganese superoxide dismutase (MnSOD or SOD2), and extracellularsuperoxide dismutase (ECSOD or SOD3). SOD1 is a 32 kDa homodimer of a153-residue polypeptide with one copper- and one zinc-binding site persubunit, which is encoded by the SOD1 gene (GeneBank access No.:NM_000454.4) on human chromosome 21 (see Table 2). SOD1 catalyzes thereaction of superoxide anion (O²⁻) into molecular oxygen (O₂) andhydrogen peroxide (H₂O₂) at a bound copper ion. The intracellularconcentration of SOD1 is high (ranging from 10 to 100 μM), accountingfor 1% of the total protein content in the central nervous system (CNS).The protein is localized not only in the cytoplasm but also in thenucleus, lysosomes, peroxisomes, and mitochondrial intermembrane spacesin eukaryotic cells (Lindenau J et al., Glia, 2000, 29, 25-34).

Mutations in the SOD1 gene are carried by 15-20% of fALS patients and by1-2% of all ALS cases. Currently, at least 170 different mutationsdistributed throughout the 153-amino acid SOD1 polypeptide have beenfound to cause ALS, and an updated list can be found at the ALS onlineGenetic Database (ALSOD) (Wroe R et al., Amyotroph Lateral Scler., 2008,9, 249-250). Table 1 lists some examples of mutations in SOD1 in ALS.These mutations are predominantly single amino acid substitutions (i.e.missense mutations) although deletions, insertions, and C-terminaltruncations also occur. Different SOD1 mutations display differentgeographic distribution patterns. For instance, 40-50% of all Americanswith ALS caused by SOD1 gene mutations have a particular mutationAla4Val (or A4V). The A4V mutation is typically associated with moresevere signs and symptoms and the survival period is typically 2-3years. The I113T mutation is by far the most common mutation in theUnited Kingdom. The most prevalent mutation in Europe is D90A substituteand the survival period is usually greater than 10 years.

TABLE 1 Examples of SOD1 mutations in ALS Location Mutations Exon1 (220bp) Q22L; E21K, G; F20C; N19S; G16A, S; V14M, S; G12R; G10G, V, R; L8Q,V; V7E; C6G, F; V5L; A4T, V, S Exon2 (97 bp) T54R; E49K; H48R, Q; V47F,A; H46R; F45C; H43R; G41S, D; G37R; V29, insA Exon3 (70 bp) D76Y, V;G72S, C; L67R; P66A; N65S; S59I, S Exon4 (118 bp) D124G, V; V118L,InsAAAAC; L117V; T116T; R115G; G114A; I113T, F; I112M, T; G108V; L106V,F; S106L, delTCACTC; I104F; D101G, Y, H, N; E100G, K; I99V; V97L, M;D96N, V; A95T, V; G935, V, A, C, R, D; D90V, A; A89T, V;T88delACTGCTGAC; V87A, M; N86I, S, D, K; G85R, S; L84V, F; H80R Exon5(461 bp) I151T, S; I149T; V148I, G; G147D, R; C146R, stop; A145T, G;L144F, S; G141E, stop; A140A, G; N139D, K, H, N; G138E; T137R; S134N;E133V, delGAA, insTT; E132insTT; G127R, InsTGGG; L126S, delITT, stop;D126, delTT

To investigate the mechanism of neuronal death associated with SOD1 genedefects, several rodent models of SOD1-linked ALS were developed in theart, which express the human SOD1 gene with different mutations,including missense mutations, small deletions, or insertions.Non-limiting examples of ALS mouse models include SOD1^(G93A),SOD1^(A4V), SOD1^(G37R), SOD1^(G85R), SOD1^(D90A), SOD1^(L84V),SOD1^(I113T), SOD1^(H36R/H48Q), SOD1^(G127X), SOD1^(L126X), andSOD1^(L126deITT). There are two transgenic rat models carrying twodifferent human SOD1 mutations: SOD1^(H46R) and SOD1^(G93R). Theserodent ALS models can develop muscle weakness similar to human ALSpatients and other pathogenic features that reflect severalcharacteristics of the human disease, in particular, the selective deathof spinal motor neurons, aggregation of protein inclusions in motorneurons and microglial activation. It is well known in the art that thetransgenic rodents are good models of human SOD1-associated ALS diseaseand provide models for studying disease pathogenesis and developingdisease treatment.

Studies in animal and cellular models showed that SOD1 pathogenicvariants cause ALS by gain of function. That is to say, the superoxidedismutase enzyme gains new but harmful properties when altered by SOD1mutations. For example, some SOD1 mutated variants in ALS increaseoxidative stress (e.g., increased accumulation of toxic superoxideradicals) by disrupting the redox cycle. Other studies also indicatethat some SOD1 mutated variants in ALS might acquire toxic propertiesthat are independent of its normal physiological function (such asabnormal aggregation of misfolded SOD1 variants. In the aberrant redoxchemistry model, mutant SOD1 is unstable and through aberrant chemistryinteracts with nonconventional substrates causing overproduction ofreactive oxygen species (ROS). In the protein toxicity model, unstable,misfolded SOD1 aggregates into cytoplasmic inclusion bodies,sequestering proteins crucial for cellular processes. These twohypotheses are not mutually exclusive. It has been shown that oxidationof selected histidine residues that bind metals in the active sitemediates SOD1 aggregation.

The aggregated mutant SOD1 protein may also induce mitochondrialdysfunction (Vehvilainen P et al., Front Cell Neurosci., 2014, 8, 126),impairment of axonal transport, aberrant RNA metabolism, glial cellpathology and glutamate excitotoxicity. In some sporadic ALS cases,misfolded wild-type SOD1 protein is found in diseased motor neuronswhich forms a “toxic conformation” that is similar to that which is seenwith familial ALS-linked SOD1 variants (Rotunno M S and Bosco D A, FrontCell Neurosci., 2013, 16, 7, 253). Such evidence suggests that ALS is aprotein folding diseases analogous to other neurodegenerative diseasessuch as Alzheimer's disease and Parkinson's disease.

Currently, no curative treatments are available for patients sufferingfrom ALS. The only FDA approved drug Riluzole, an inhibitor of glutamaterelease, has a moderate effect on ALS, only extending survival by 2-3months if it is taken for 18 months. Unfortunately, patients takingriluzole do not experience any slowing in disease progression orimprovement in muscle function. Therefore, riluzole does not present acure, or even an effective treatment. Researchers continue to search forbetter therapeutic agents.

Therapeutic approaches that may prevent or ameliorate SOD1 aggregationhave been tested previously. For example, arimoclomol, a hydroxylaminederivative, is a drug that targets heat shock proteins, which arecellular defense mechanisms against these aggregates. Studiesdemonstrated that treatment with arimoclomol improved muscle function inSOD1 mouse models. Other drugs that target one or more cellular defectsin ALS may include AMPA antagonists such as talampanel, beta-lactamantibiotics, which may reduce glutamate-induced excitotoxicity to motorneurons; Bromocriptine that may inhibit oxidative induced motor neurondeath (e.g. U.S. Patent publication No. 20110105517; the content ofwhich is incorporated herein by reference in its entirety);1,3-diphenylurea derivative or multikinase inhibitor which may reduceSOD1 gene expression (e.g., U.S. Patent Publication No. 20130225642; thecontent of which is incorporated herein by reference in its entirety);dopamine agonist pramipexole and its anantiomer dexpramipexole, whichmay ameliorate the oxidative response in mitochondria; nimesulide, whichinhibits cyclooxygenase enzyme (e.g., U.S. Patent Publication No.20060041022; the content of which is incorporated herein by reference inits entirety); drugs that act as free radical scavengers (e.g. U.S. Pat.No. 6,933,310 and PCT Patent Publication No.: WO2006075434; the contentof each of which is incorporated herein by reference in their entirety).

Another approach to inhibit abnormal SOD1 protein aggregation is tosilence/inhibit SOD1 gene expression in ALS. It has been reported thatsmall interfering RNAs for specific gene silencing of the mutated alleleare therapeutically beneficial for the treatment of fALS (e.g., Ralgh GS et al., Nat. Medicine, 2005, 11(4), 429-433; and Raoul C et al., Nat.Medicine, 2005, 11(4), 423-428; and Maxwell M M et al., PNAS, 2004,101(9), 3178-3183; and Ding H et al., Chinese Medical J., 2011, 124(1),106-110; and Scharz D S et al., Plos Genet., 2006, 2(9), e140; thecontent of each of which is incorporated herein by reference in theirentirety).

Many other RNA therapeutic agents that target the SOD1 gene and modulateSOD1 expression in ALS are taught in the art. Such RNA based agentsinclude antisense oligonucleotides and double stranded small interferingRNAs. See, e.g., Wang H et al., J Biol. Chem., 2008, 283(23),15845-15852); U.S. Pat. Nos. 7,498,316; 7,632,938; 7,678,895; 7,951,784;7,977,314; 8,183,219; 8,309,533 and 8, 586, 554; and U.S. Patentpublication Nos. 2006/0229268 and 2011/0263680; the content of each ofwhich is herein incorporated by reference in their entirety.

The present invention provides modulatory polynucleotides, e.g., siRNAmolecules targeting the SOD1 gene and methods for their design andmanufacture. Particularly, the present invention employs viral vectorssuch as adeno-associated viral (AAV) vectors comprising the nucleic acidsequence encoding the siRNA molecules of the present invention. The AAVvectors comprising the nucleic acid sequence encoding the siRNAmolecules of the present invention may increase the delivery of activeagents into motor neurons. The siRNA duplexes or encoding dsRNAtargeting the SOD1 gene may be able to inhibit SOD1 gene expression(e.g., mRNA level) significantly inside cells; therefore, amelioratingSOD1 expression induced stress inside the cells such as aggregation ofprotein and formation of inclusions, increased free radicals,mitochondrial dysfunction, and RNA metabolism.

Such siRNA mediated SOD1 expression inhibition may be used for treatingALS. According to the present invention, methods for treating and/orameliorating ALS in a patient comprises administering to the patient aneffective amount of AAV vector comprising a nucleic acid sequenceencoding the siRNA molecules of the present invention into cells. Theadministration of the AAV vector comprising such a nucleic acid sequencewill encode the siRNA molecules which cause the inhibition/silence ofSOD1 gene expression.

In one embodiment, the vectors, e.g., AAV encoding the modulatorypolynucleotide, reduce the expression of mutant SOD1 in a subject. Thereduction of mutant SOD1 can also reduce the formation of toxicaggregates which can cause mechanisms of toxicity such as, but notlimited to, oxidative stress, mitochondrial dysfunction, impaired axonaltransport, aberrant RNA metabolism, glial cell pathology and/orglutamate excitotoxicity.

In one embodiment, the vector, e.g., AAV vectors, reduces the amount ofSOD1 in a subject in need thereof and thus provides a therapeuticbenefit as described herein.

Compositions of the Invention

siRNA Molecules

The present invention relates to RNA interference (RNAi) inducedinhibition of gene expression for treating neurodegenerative disorders.Provided herein are siRNA duplexes or encoded dsRNA that target the SOD1gene (referred to herein collectively as “siRNA molecules”). Such siRNAduplexes or encoded dsRNA can reduce or silence SOD1 gene expression incells, for example, motor neurons, thereby, ameliorating symptoms of ALSsuch as, but not limited to, motor neuron death and muscle atrophy.

RNAi (also known as post-transcriptional gene silencing (PTGS),quelling, or co-suppression) is a post-transcriptional gene silencingprocess in which RNA molecules, in a sequence specific manner, inhibitgene expression, typically by causing the destruction of specific mRNAmolecules. The active components of RNAi are short/small double strandedRNAs (dsRNAs), called small interfering RNAs (siRNAs), that typicallycontain 15-30 nucleotides (e.g., 19 to 25, 19 to 24 or 19-21nucleotides) and 2 nucleotide 3′ overhangs and that match the nucleicacid sequence of the target gene. These short RNA species may benaturally produced in vivo by Dicer-mediated cleavage of larger dsRNAsand they are functional in mammalian cells.

Naturally expressed small RNA molecules, named microRNAs (miRNAs),elicit gene silencing by regulating the expression of mRNAs. The miRNAscontaining RNA Induced Silencing Complex (RISC) targets mRNAs presentinga perfect sequence complementarity with nucleotides 2-7 in the 5′ regionof the miRNA which is called the seed region, and other base pairs withits 3′ region. miRNA mediated down regulation of gene expression may becaused by cleavage of the target mRNAs, translational inhibition of thetarget mRNAs, or mRNA decay. miRNA targeting sequences are usuallylocated in the 3′-UTR of the target mRNAs. A single miRNA may targetmore than 100 transcripts from various genes, and one mRNA may betargeted by different miRNAs.

siRNA duplexes or dsRNA targeting a specific mRNA may be designed andsynthesized in vitro and introduced into cells for activating RNAiprocesses. Elbashir et al. demonstrated that 21-nucleotide siRNAduplexes (termed small interfering RNAs) were capable of effectingpotent and specific gene knockdown without inducing immune response inmammalian cells (Elbashir S M et al., Nature, 2001, 411, 494-498). Sincethis initial report, post-transcriptional gene silencing by siRNAsquickly emerged as a powerful tool for genetic analysis in mammaliancells and has the potential to produce novel therapeutics.

In vitro synthetized siRNA molecules may be introduced into cells inorder to activate RNAi. An exogenous siRNA duplex, when it is introducedinto cells, similar to the endogenous dsRNAs, can be assembled to formthe RNA Induced Silencing Complex (RISC), a multiunit complex thatfacilitates searching through the genome for RNA sequences that arecomplementary to one of the two strands of the siRNA duplex (i.e., theantisense strand). During the process, the sense strand (or passengerstrand) of the siRNA is lost from the complex, while the antisensestrand (or guide strand) of the siRNA is matched with its complementaryRNA. In particular, the targets of siRNA containing RISC complex aremRNAs presenting a perfect sequence complementarity. Then, siRNAmediated gene silencing occurs, cleaving, releasing and degrading thetarget.

The siRNA duplex comprised of a sense strand homologous to the targetmRNA and an antisense strand that is complementary to the target mRNAoffers much more advantage in terms of efficiency for target RNAdestruction compared to the use of the single strand (ss)-siRNAs (e.g.antisense strand RNA or antisense oligonucleotides). In many cases itrequires higher concentration of the ss-siRNA to achieve the effectivegene silencing potency of the corresponding duplex.

Any of the foregoing molecules may be encoded by an AAV vector or vectorgenome.

Design and Sequences of siRNA Duplexes Targeting SOD1 Gene

Some guidelines for designing siRNAs have been proposed in the art.These guidelines generally recommend generating a 19-nucleotide duplexedregion, symmetric 2-3 nucleotide 3′ overhangs, 5-phosphate and3-hydroxyl groups targeting a region in the gene to be silenced. Otherrules that may govern siRNA sequence preference include, but are notlimited to, (i) A/U at the 5′ end of the antisense strand; (ii) G/C atthe 5′ end of the sense strand; (iii) at least five A/U residues in the5′ terminal one-third of the antisense strand; and (iv) the absence ofany GC stretch of more than 9 nucleotides in length. In accordance withsuch consideration, together with the specific sequence of a targetgene, highly effective siRNA molecules essential for suppressingmammalian target gene expression may be readily designed.

According to the present invention, siRNA molecules (e.g., siRNAduplexes or encoded dsRNA) that target the human SOD1 gene are designed.Such siRNA molecules can specifically, suppress SOD1 gene expression andprotein production. In some aspects, the siRNA molecules are designedand used to selectively “knock out” SOD1 gene variants in cells, i.e.,mutated SOD1 transcripts that are identified in patients with ALSdisease (e.g., mutations in Table 1). In some aspects, the siRNAmolecules are designed and used to selectively “knock down” SOD1 genevariants in cells. In other aspects, the siRNA molecules are able toinhibit or suppress both wild type and mutated alleles of SOD1 geneirrelevant of any particular mutations in the SOD1 gene.

In one embodiment, an siRNA molecule of the present invention comprisesa sense strand and a complementary antisense strand in which bothstrands are hybridized together to form a duplex structure. Theantisense strand has sufficient complementarity to the SOD1 mRNAsequence to direct target-specific RNAi, i.e., the siRNA molecule has asequence sufficient to trigger the destruction of the target mRNA by theRNAi machinery or process.

In some embodiments, the antisense strand and target mRNA sequences are100% complementary. The antisense strand may be complementary to anypart of the target mRNA sequence.

In other embodiments, the antisense strand and target mRNA sequencescomprise at least one mismatch. As a non-limiting example, the antisensestrand and the target mRNA sequence are at least 30%, 40%, 50%, 60%,70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% or at least 20-30%, 20-40%, 20-50%,20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-99%, 30-40%, 30-50%, 30-60%,30-70%, 30-80%, 30-90%, 30-95%, 30-99%, 40-50%, 40-60%, 40-70%, 40-80%,40-90%, 40-95%, 40-99%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-99%,60-70%, 60-80%, 60-90%, 60-95%, 60-99%, 70-80%, 70-90%, 70-95%, 70-99%,80-90%, 80-95%, 80-99%, 90-95%, 90-99% or 95-99% complementary.

According to the present invention, the siRNA molecule has a length fromabout 10-50 or more nucleotides, i.e., each strand comprising 10-50nucleotides (or nucleotide analogs). Preferably, the siRNA molecule hasa length from about 15-30, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of thestrands is sufficiently complementary to a target region. In oneembodiment, the siRNA molecule has a length from about 19 to 25, 19 to24 or 19 to 21 nucleotides.

In some embodiments, the siRNA molecules of the present invention can besynthetic RNA duplexes comprising about 19 nucleotides to about 25nucleotides, and two overhanging nucleotides at the 3′-end. In someaspects, the siRNA molecules may be unmodified RNA molecules. In otheraspects, the siRNA molecules may contain at least one modifiednucleotide, such as base, sugar or backbone modifications.

In other embodiments, the siRNA molecules of the present invention canbe encoded in plasmid vectors, viral vectors (e.g., AAV vectors), genomeor other nucleic acid expression vectors for delivery to a cell.

DNA expression plasmids can be used to stably express the siRNA duplexesor dsRNA of the present invention in cells and achieve long-terminhibition of the target gene. In one aspect, the sense and antisensestrands of a siRNA duplex are typically linked by a short spacersequence leading to the expression of a stem-loop structure termed shorthairpin RNA (shRNA). The hairpin is recognized and cleaved by Dicer,thus generating mature siRNA molecules.

According to the present invention, AAV vectors comprising the nucleicacids encoding the siRNA molecules targeting SOD1 mRNA are produced, theAAV vector serotypes may be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrh10,AAV-DJ8 and AAV-DJ, and variants thereof.

In some embodiments, the siRNA duplexes or encoded dsRNA of the presentinvention suppress (or degrade) target mRNA (i.e., SOD1). Accordingly,the siRNA duplexes or encoded dsRNA can be used to substantially inhibitSOD1 gene expression in a cell, for example a motor neuron. In someaspects, the inhibition of SOD1 gene expression refers to an inhibitionby at least about 20%, preferably by at least about 30%, 40%, 50%, 60%,70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%,20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%,30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%,40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%,50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%,70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100%.Accordingly, the protein product of the targeted gene may be inhibitedby at least about 20%, preferably by at least about 30%, 40%, 50%, 60%,70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%,20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%,30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%,40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%,50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%,70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100%.The SOD1 gene can be either a wild type gene or a mutated SOD1 gene withat least one mutation. Accordingly, the protein is either wild typeprotein or a mutated polypeptide with at least one mutation.

According to the present invention, siRNA duplexes or encoded dsRNAtargeting human SOD1 gene were designed and tested for their ability inreducing SOD1 mRNA levels in cultured cells. Such siRNA duplexes includethose listed in Table 3. As a non-limiting example, the siRNA duplexesmay be siRNA duplex IDs: D-2757, D-2806, D-2860, D-2861, D-2875, D-2871,D-2758, D-2759, D-2866, D-2870, D-2823 and D-2858.

In one embodiment, the 3′ stem arm of the siRNA duplexes or encodeddsRNA targeting the human SOD1 gene may have 11 nucleotides downstreamof the 3′ end of the guide strand which have complementarity to the 11of the 13 nucleotides upstream of the 5′ end of the passenger strand inthe 5′ stem arm.

In one embodiment, the siRNA duplexes or encoded dsRNA targeting humanSOD1 gene may have a cysteine which is 6 nucleotides downstream of the3′ end of the 3′ stem arm of the modulatory polynucleotide.

In one embodiment, the siRNA duplexes or encoded dsRNA targeting humanSOD1 gene comprise a miRNA seed match for the guide strand. In anotherembodiment, the siRNA duplexes or encoded dsRNA targeting human SOD1gene comprise a miRNA seed match for the passenger strand. In yetanother embodiment, the siRNA duplexes or encoded dsRNA targeting humanSOD1 gene do not comprise a seed match for the guide or passengerstrand.

In one embodiment, the siRNA duplexes or encoded dsRNA targeting humanSOD1 gene may have almost no significant full-length off targets for theguide strand. In another embodiment, the siRNA duplexes or encoded dsRNAtargeting human SOD1 gene may have almost no significant full-length offtargets for the passenger strand. The siRNA duplexes or encoded dsRNAtargeting human SOD1 gene may have less than 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 1-5%, 2-6%, 3-7%, 4-8%, 5-9%, 5-10% 6-10% full-length offtargets for the passenger strand. In yet another embodiment, the siRNAduplexes or encoded dsRNA targeting human SOD1 gene may have almost nosignificant full-length off targets for the guide strand or thepassenger strand. The siRNA duplexes or encoded dsRNA targeting humanSOD1 gene may have less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,1-5%, 2-6%, 3-7%, 4-8%, 5- 9%, 5-10% 6-10% full-length off targets forthe guide or passenger strand.

In one embodiment, the siRNA duplexes or encoded dsRNA targeting humanSOD1 gene may have high activity in vitro. In another embodiment, thesiRNA duplexes or encoded dsRNA targeting the human SOD1 gene may havelow activity in vitro. In yet another embodiment, the siRNA duplexes ordsRNA targeting the human SOD1 gene may have high guide strand activityand low passenger strand activity in vitro.

In one embodiment, the siRNA duplexes or encoded dsRNA targeting thehuman SOD1 gene have a high guide strand activity and low passengerstrand activity in vitro. The target knock-down (KD) by the guide strandmay be at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5% or100%. The target knock-down by the guide strand may be 60-65%, 60-70%,60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 60-99%, 60-99.5%, 60-100%,65-70%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 65-99%, 65-99.5%,65-100%, 70-75%, 70-80%, 70-85%, 70-90%, 70-95%, 70-99%, 70-99.5%,70-100%, 75-80%, 75-85%, 75-90%, 75-95%, 75-99%, 75-99.5%, 75-100%,80-85%, 80-90%, 80-95%, 80-99%, 80-99.5%, 80-100%, 85- 90%, 85-95%,85-99%, 85-99.5%, 85-100%, 90-95%, 90-99%, 90-99.5%, 90-100%, 95-99%,95- 99.5%, 95-100%, 99-99.5%, 99-100% or 99.5-100%. As a non-limitingexample, the target knock-down (KD) by the guide strand is greater than70%.

In one embodiment, the IC₅₀ of the passenger strand for the nearest offtarget is greater than 100 multiplied by the IC₅₀ of the guide strandfor the target. As a non-limiting example, if the IC₅₀ of the passengerstrand for the nearest off target is greater than 100 multiplied by theIC₅₀ of the guide strand for the target then the siRNA duplexes orencoded dsRNA targeting the human SOD1 gene is said to have high guidestrand activity and a low passenger strand activity in vitro.

In one embodiment, the 5′ processing of the guide strand has a correctstart (n) at the 5′ end at least 75%, 80%, 85%, 90%, 95%, 99% or 100% ofthe time in vitro or in vivo. As a non-limiting example, the 5′processing of the guide strand is precise and has a correct start (n) atthe 5′ end at least 99% of the time in vitro. As a non-limiting example,the 5′ processing of the guide strand is precise and has a correct start(n) at the 5′ end at least 99% of the time in vivo.

In one embodiment, the guide-to-passenger (G:P) strand ratio expressedis 1:99, 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55,50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, 95:5, or99:1 in vitro or in vivo. As a non-limiting example, theguide-to-passenger strand ratio is 80:20 in vitro. As a non-limitingexample, the guide-to-passenger strand ratio is 80:20 in vivo.

In one embodiment, the integrity of the vector genome encoding the dsRNAis at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more than 99%of the full length of the construct.

siRNA Modification

In some embodiments, the siRNA molecules of the present invention, whennot delivered as a precursor or DNA, may be chemically modified tomodulate some features of RNA molecules, such as, but not limited to,increasing the stability of siRNAs in vivo. The chemically modifiedsiRNA molecules can be used in human therapeutic applications, and areimproved without compromising the RNAi activity of the siRNA molecules.As a non-limiting example, the siRNA molecules modified at both the 3′and the 5′ end of both the sense strand and the antisense strand.

In some aspects, the siRNA duplexes of the present invention may containone or more modified nucleotides such as, but not limited to, sugarmodified nucleotides, nucleobase modifications and/or backbonemodifications. In some aspects, the siRNA molecule may contain combinedmodifications, for example, combined nucleobase and backbonemodifications.

In one embodiment, the modified nucleotide may be a sugar-modifiednucleotide. Sugar modified nucleotides include, but are not limited to2′-fluoro, 2′-amino and 2′-thio modified ribonucleotides, e.g.,2′-fluoro modified ribonucleotides. Modified nucleotides may be modifiedon the sugar moiety, as well as nucleotides having sugars or analogsthereof that are not ribosyl. For example, the sugar moieties may be, orbe based on, mannoses, arabinoses, glucopyranoses, galactopyranoses,4′-thioribose, and other sugars, heterocycles, or carbocycles.

In one embodiment, the modified nucleotide may be a nucleobase-modifiednucleotide.

In one embodiment, the modified nucleotide may be a backbone-modifiednucleotide. In some embodiments, the siRNA duplexes of the presentinvention may further comprise other modifications on the backbone. Anormal “backbone”, as used herein, refers to the repeatingly alternatingsugar-phosphate sequences in a DNA or RNA molecule. Thedeoxyribose/ribose sugars are joined at both the 3′-hydroxyl and5′-hydroxyl groups to phosphate groups in ester links, also known as“phosphodiester” bonds/linker (PO linkage). The PO backbones may bemodified as “phosphorothioate backbone (PS linkage). In some cases, thenatural phosphodiester bonds may be replaced by amide bonds but the fouratoms between two sugar units are kept. Such amide modifications canfacilitate the solid phase synthesis of oligonucleotides and increasethe thermodynamic stability of a duplex formed with siRNA complement.See e.g., Mesmaeker et al., Pure & Appl. Chem., 1997, 3, 437-440; thecontent of which is incorporated herein by reference in its entirety.

Modified bases refer to nucleotide bases such as, for example, adenine,guanine, cytosine, thymine, uracil, xanthine, inosine, and queuosinethat have been modified by the replacement or addition of one or moreatoms or groups. Some examples of modifications on the nucleobasemoieties include, but are not limited to, alkylated, halogenated,thiolated, aminated, amidated, or acetylated bases, individually or incombination. More specific examples include, for example,5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine,N,N,-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine,1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine andother nucleotides having a modification at the 5 position,5-(2-amino)propyl uridine, 5-halocytidine, 5-halouridine,4-acetylcytidine, 1-methyladenosine, 2-methyladenosine,3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine,2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine,deazanucleotides such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine,6-azothymidine, 5-methyl-2-thiouridine, other thio bases such as2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine,pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthylgroups, any 0- and N-alkylated purines and pyrimidines such asN6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyaceticacid, pyridine-4-one, pyridine-2-one, phenyl and modified phenyl groupssuch as aminophenol or 2,4,6-trimethoxy benzene, modified cytosines thatact as G-clamp nucleotides, 8-substituted adenines and guanines,5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkylnucleotides, carboxyalkylaminoalkyl nucleotides, andalkylcarbonylalkylated nucleotides.

In one embodiment, the modified nucleotides may be on just the sensestrand.

In another embodiment, the modified nucleotides may be on just theantisense strand.

In some embodiments, the modified nucleotides may be in both the senseand antisense strands.

In some embodiments, the chemically modified nucleotide does not affectthe ability of the antisense strand to pair with the target mRNAsequence, such as the SOD1 mRNA sequence.

Vectors

In some embodiments, the siRNA molecules described herein can be encodedby vectors such as plasmids or viral vectors. In one embodiment, thesiRNA molecules are encoded by viral vectors. Viral vectors may be, butare not limited to, Herpesvirus (HSV) vectors, retroviral vectors,adenoviral vectors, adeno-associated viral vectors, lentiviral vectors,and the like. In some specific embodiments, the viral vectors are AAVvectors.

Retroviral Vectors

In some embodiments, the siRNA duplex targeting SOD1 gene may be encodedby a retroviral vector (See, e.g., U.S. Pat. Nos. 5,399,346; 5,124,263;4,650,764 and 4,980,289; the content of each of which is incorporatedherein by reference in their entirety).

Adenoviral Vectors

Adenoviruses are eukaryotic DNA viruses that can be modified toefficiently deliver a nucleic acid to a variety of cell types in vivo,and have been used extensively in gene therapy protocols, including fortargeting genes to neural cells. Various replication defectiveadenovirus and minimum adenovirus vectors have been described fornucleic acid therapeutics (See, e.g., PCT Patent Publication Nos.WO199426914, WO 199502697, WO199428152, WO199412649, WO199502697 andWO199622378; the content of each of which is incorporated by referencein their entirety). Such adenoviral vectors may also be used to deliversiRNA molecules of the present invention to cells.

Adeno-Associated Viral (AAV) Vectors

An adeno-associated virus (AAV) is a dependent parvovirus (like otherparvoviruses) which is a single stranded non-enveloped DNA virus havinga genome of about 5000 nucleotides in length and which contains two openreading frames encoding the proteins responsible for replication (Rep)and the structural protein of the capsid (Cap). The open reading framesare flanked by two Inverted Terminal Repeat (ITR) sequences, which serveas the origin of replication of the viral genome. Furthermore, the AAVgenome contains a packaging sequence, allowing packaging of the viralgenome into an AAV capsid. The AAV vector requires a co-helper (e.g.,adenovirus) to undergo productive infection in infected cells. In theabsence of such helper functions, the AAV virions essentially enter hostcells and integrate into the cells' genome.

AAV vectors have been investigated for siRNA delivery because of severalunique features. Non-limiting examples of the features include (i) theability to infect both dividing and non-dividing cells; (ii) a broadhost range for infectivity, including human cells; (iii) wild-type AAVhas not been associated with any disease and has not been shown toreplicate in infected cells; (iv) the lack of cell-mediated immuneresponse against the vector and (v) the non-integrative nature in a hostchromosome thereby reducing potential for long-term expression.Moreover, infection with AAV vectors has minimal influence on changingthe pattern of cellular gene expression (Stilwell and Samulski et al.,Biotechniques, 2003, 34, 148).

Typically, AAV vectors for siRNA delivery may be recombinant viralvectors which are replication defective as they lack sequences encodingfunctional Rep and Cap proteins within the viral genome. In some cases,the defective AAV vectors may lack most or all coding sequences andessentially only contains one or two AAV ITR sequences and a packagingsequence.

AAV vectors may also comprise self-complementary AAV vectors (scAAVs).scAAV vectors contain both DNA strands which anneal together to formdouble stranded DNA. By skipping second strand synthesis, scAAVs allowfor rapid expression in the cell.

In one embodiment, the AAV vector used in the present invention is ascAAV.

In one embodiment, the AAV vector used in the present invention is anssAAV.

Methods for producing and/or modifying AAV vectors are disclosed in theart such as pseudotyped AAV vectors (PCT Patent Publication Nos.WO200028004; WO200123001; WO2004112727; WO 2005005610 and WO 2005072364,the content of each of which is incorporated herein by reference intheir entirety).

AAV vectors comprising the nucleic acid sequence for the siRNA moleculesmay be prepared or derived from various serotypes of AAVs, including,but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ8and AAV-DJ. In some cases, different serotypes of AAVs may be mixedtogether or with other types of viruses to produce chimeric AAV vectors.

In one embodiment, the AAV vectors comprising a nucleic acid sequenceencoding the siRNA molecules of the present invention may be introducedinto mammalian cells.

AAV vectors may be modified to enhance the efficiency of delivery. Suchmodified AAV vectors comprising the nucleic acid sequence encoding thesiRNA molecules of the present invention can be packaged efficiently andcan be used to successfully infect the target cells at high frequencyand with minimal toxicity.

In some embodiments, the AAV vector comprising a nucleic acid sequenceencoding the siRNA molecules of the present invention may be a humanserotype AAV vector. Such human AAV vector may be derived from any knownserotype, e.g., from any one of serotypes AAV1-AAV11. As non-limitingexamples, AAV vectors may be vectors comprising an AAV1-derived genomein an AAV1-derived capsid; vectors comprising an AAV2-derived genome inan AAV2-derived genome; vectors comprising an AAV4-derived genome in anAAV4 derived capsid; vectors comprising an AAV6-derived genome in anAAV6 derived capsid or vectors comprising an AAV9-derived genome in anAAV9 derived capsid.

In other embodiments, the AAV vector comprising a nucleic acid sequencefor encoding siRNA molecules of the present invention may be apseudotyped hybrid or chimeric AAV vector which contains sequencesand/or components originating from at least two different AAV serotypes.Pseudotyped AAV vectors may be vectors comprising an AAV genome derivedfrom one AAV serotype and a capsid protein derived at least in part froma different AAV serotype. As non-limiting examples, such pseudotyped AAVvectors may be vectors comprising an AAV2-derived genome in anAAV1-derived capsid; or vectors comprising an AAV2-derived genome in anAAV6-derived capsid; or vectors comprising an AAV2-derived genome in anAAV4-derived capsid; or an AAV2-derived genome in an AAV9-derivedcapsid. In like fashion, the present invention contemplates any hybridor chimeric AAV vector.

In other embodiments, AAV vectors comprising a nucleic acid sequenceencoding the siRNA molecules of the present invention may be used todeliver siRNA molecules to the central nervous system (e.g., U.S. Pat.No. 6,180,613; the contents of which is herein incorporated by referencein its entirety).

In some aspects, the AAV vectors comprising a nucleic acid sequenceencoding the siRNA molecules of the present invention may furthercomprise a modified capsid including peptides from non-viral origin. Inother aspects, the AAV vector may contain a CNS specific chimeric capsidto facilitate the delivery of encoded siRNA duplexes into the brain andthe spinal cord. For example, an alignment of cap nucleotide sequencesfrom AAV variants exhibiting CNS tropism may be constructed to identifyvariable region (VR) sequence and structure.

In one embodiment, the AAV vector comprising a nucleic acid sequenceencoding the siRNA molecules of the present invention may encode siRNAmolecules which are polycistronic molecules. The siRNA molecules mayadditionally comprise one or more linkers between regions of the siRNAmolecules.

In one embodiment, the encoded siRNA molecule may be located downstreamof a promoter in an expression vector such as, but not limited to, CMV,U6, CBA or a CBA promoter with a SV40 intron. Further, the encoded siRNAmolecule may also be located upstream of the polyadenylation sequence inan expression vector. As a non-limiting example, the encoded siRNAmolecule may be located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30or more than 30 nucleotides downstream from the promoter and/or upstreamof the polyadenylation sequence in an expression vector. As anothernon-limiting example, the encoded siRNA molecule may be located within1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15,10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 20-25, 20-30 or 25-30nucleotides downstream from the promoter and/or upstream of thepolyadenylation sequence in an expression vector. As a non-limitingexample, the encoded siRNA molecule may be located within the first 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or more than 25% ofthe nucleotides downstream from the promoter and/or upstream of thepolyadenylation sequence in an expression vector. As anothernon-limiting example, the encoded siRNA molecule may be located with thefirst 1-5%, 1-10%, 1-15%, 1-20%, 1-25%, 5-10%, 5-15%, 5-20%, 5-25%,10-15%, 10-20%, 10-25%, 15-20%, 15-25%, or 20-25% downstream from thepromoter and/or upstream of the polyadenylation sequence in anexpression vector.

In one embodiment, the encoded siRNA molecule may be located upstream ofthe polyadenylation sequence in an expression vector. Further, theencoded siRNA molecule may be located downstream of a promoter such as,but not limited to, CMV, U6, CBA or a CBA promoter with a SV40 intron inan expression vector. As a non-limiting example, the encoded siRNAmolecule may be located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30or more than 30 nucleotides downstream from the promoter and/or upstreamof the polyadenylation sequence in an expression vector. As anothernon-limiting example, the encoded siRNA molecule may be located within1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15,10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 20-25, 20-30 or 25-30nucleotides downstream from the promoter and/or upstream of thepolyadenylation sequence in an expression vector. As a non-limitingexample, the encoded siRNA molecule may be located within the first 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or more than 25% ofthe nucleotides downstream from the promoter and/or upstream of thepolyadenylation sequence in an expression vector. As anothernon-limiting example, the encoded siRNA molecule may be located with thefirst 1-5%, 1-10%, 1-15%, 1-20%, 1-25%, 5-10%, 5-15%, 5-20%, 5-25%,10-15%, 10-20%, 10-25%, 15-20%, 15-25%, or 20-25% downstream from thepromoter and/or upstream of the polyadenylation sequence in anexpression vector.

In one embodiment, the encoded siRNA molecule may be located in a scAAV.

In one embodiment, the encoded siRNA molecule may be located in anssAAV.

In one embodiment, the encoded siRNA molecule may be located near the 5′end of the flip ITR in an expression vector. In another embodiment, theencoded siRNA molecule may be located near the 3′ end of the flip ITR inan expression vector. In yet another embodiment, the encoded siRNAmolecule may be located near the 5′ end of the flop ITR in an expressionvector. In yet another embodiment, the encoded siRNA molecule may belocated near the 3′ end of the flop ITR in an expression vector. In oneembodiment, the encoded siRNA molecule may be located between the 5′ endof the flip ITR and the 3′ end of the flop ITR in an expression vector.In one embodiment, the encoded siRNA molecule may be located between(e.g., half-way between the 5′ end of the flip ITR and 3′ end of theflop ITR or the 3′ end of the flop ITR and the 5′ end of the flip ITR),the 3′ end of the flip ITR and the 5′ end of the flip ITR in anexpression vector. As a non-limiting example, the encoded siRNA moleculemay be located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more than30 nucleotides downstream from the 5′ or 3′ end of an ITR (e.g., Flip orFlop ITR) in an expression vector. As a non-limiting example, theencoded siRNA molecule may be located within 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30 or more than 30 nucleotides upstream from the 5′ or 3′ end ofan ITR (e.g., Flip or Flop ITR) in an expression vector. As anothernon-limiting example, the encoded siRNA molecule may be located within1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15,10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 20-25, 20-30 or 25-30nucleotides downstream from the 5′ or 3′ end of an ITR (e.g., Flip orFlop ITR) in an expression vector. As another non-limiting example, theencoded siRNA molecule may be located within 1-5, 1-10, 1-15, 1-20,1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30,15-20, 15-25, 15-30, 20-25, 20-30 or 25-30 upstream from the 5′ or 3′end of an ITR (e.g., Flip or Flop ITR) in an expression vector. As anon-limiting example, the encoded siRNA molecule may be located withinthe first 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or morethan 25% of the nucleotides upstream from the 5′ or 3′ end of an ITR(e.g., Flip or Flop ITR) in an expression vector. As anothernon-limiting example, the encoded siRNA molecule may be located with thefirst 1-5%, 1-10%, 1-15%, 1-20%, 1-25%, 5-10%, 5-15%, 5-20%, 5-25%,10-15%, 10-20%, 10-25%, 15-20%, 15-25%, or 20-25% downstream from the 5′or 3′ end of an ITR (e.g., Flip or Flop ITR) in an expression vector.

Expression Vector

In one embodiment, an expression vector (e.g., AAV vector) may compriseat least one of the modulatory polynucleotides comprising at least oneof the expression vectors described herein.

In one embodiment, an expression vector may comprise, from ITR to ITRrecited 5′ to 3′, an ITR, a promoter, an intron, a modulatorypolynucleotide, a polyA sequence and an ITR.

Genome Size

In one embodiment, the vector genome which comprises a nucleic acidsequence encoding the modulatory polynucleotides described herein may besingle stranded or double stranded vector genome. The size of the vectorgenome may be small, medium, large or the maximum size. Additionally,the vector genome may comprise a promoter and a polyA tail.

In one embodiment, the vector genome which comprises a nucleic acidsequence encoding the modulatory polynucleotides described herein may bea small single stranded vector genome. A small single stranded vectorgenome may be 2.7 to 3.5 kb in size such as about 2.7, 2.8, 2.9, 3.0,3.1, 3.2, 3.3, 3.4, and 3.5 kb in size. As a non-limiting example, thesmall single stranded vector genome may be 3.2 kb in size. Additionally,the vector genome may comprise a promoter and a polyA tail.

In one embodiment, the vector genome which comprises a nucleic acidsequence encoding the modulatory polynucleotides described herein may bea small double stranded vector genome. A small double stranded vectorgenome may be 1.3 to 1.7 kb in size such as about 1.3, 1.4, 1.5, 1.6,and 1.7 kb in size. As a non-limiting example, the small double strandedvector genome may be 1.6 kb in size. Additionally, the vector genome maycomprise a promoter and a polyA tail.

In one embodiment, the vector genome which comprises a nucleic acidsequence encoding the modulatory polynucleotides described herein e.g.,siRNA or dsRNA, may be a medium single stranded vector genome. A mediumsingle stranded vector genome may be 3.6 to 4.3 kb in size such as about3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2 and 4.3 kb in size. As a non-limitingexample, the medium single stranded vector genome may be 4.0 kb in size.Additionally, the vector genome may comprise a promoter and a polyAtail.

In one embodiment, the vector genome which comprises a nucleic acidsequence encoding the modulatory polynucleotides described herein may bea medium double stranded vector genome. A medium double stranded vectorgenome may be 1.8 to 2.1 kb in size such as about 1.8, 1.9, 2.0, and 2.1kb in size. As a non-limiting example, the medium double stranded vectorgenome may be 2.0 kb in size. Additionally, the vector genome maycomprise a promoter and a polyA tail.

In one embodiment, the vector genome which comprises a nucleic acidsequence encoding the modulatory polynucleotides described herein may bea large single stranded vector genome. A large single stranded vectorgenome may be 4.4 to 6.0 kb in size such as about 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 and 6.0 kb insize. As a non-limiting example, the large single stranded vector genomemay be 4.7 kb in size. As another non-limiting example, the large singlestranded vector genome may be 4.8 kb in size. As yet anothernon-limiting example, the large single stranded vector genome may be 6.0kb in size. Additionally, the vector genome may comprise a promoter anda polyA tail.

In one embodiment, the vector genome which comprises a nucleic acidsequence encoding the modulatory polynucleotides described herein may bea large double stranded vector genome. A large double stranded vectorgenome may be 2.2 to 3.0 kb in size such as about 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9 and 3.0 kb in size. As a non-limiting example, thelarge double stranded vector genome may be 2.4 kb in size. Additionally,the vector genome may comprise a promoter and a polyA tail.

Promoters

A person skilled in the art may recognize that a target cell may requirea specific promoter including but not limited to a promoter that isspecies specific, inducible, tissue-specific, or cell cycle-specificParr et al., Nat. Med. 3:1145-9 (1997); the contents of which are hereinincorporated by reference in its entirety).

In one embodiment, the promoter is a promoter deemed to be efficient todrive the expression of the modulatory polynucleotide.

In one embodiment, the promoter is a promoter having a tropism for thecell being targeted.

In one embodiment, the promoter is a weak promoter which providesexpression of a payload e.g., a modulatory polynucleotide, e.g., siRNAor dsRNA, for a period of time in targeted tissues such as, but notlimited to, nervous system tissues. Expression may be for a period of 1hour, 2, hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17days, 18 days, 19 days, 20 days, 3 weeks, 22 days, 23 days, 24 days, 25days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months,16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years,8 years, 9 years, 10 years or more than 10 years. Expression may be for1-5 hours, 1-12 hours, 1-2 days, 1-5 days, 1-2 weeks, 1-3 weeks, 1-4weeks, 1-2 months, 1-4 months, 1-6 months, 2-6 months, 3-6 months, 3-9months, 4-8 months, 6-12 months, 1-2 years, 1-5 years, 2-5 years, 3-6years, 3-8 years, 4-8 years or 5-10 years. As a non-limiting example,the promoter is a weak promoter for sustained expression of a payload innervous tissues.

In one embodiment, the promoter may be a promoter which is less than 1kb. The promoter may have a length of 200, 210, 220, 230, 240, 250, 260,270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400,410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540,550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680,690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 or more than800. The promoter may have a length between 200-300, 200-400, 200-500,200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300-800,400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700,600-800 or 700-800.

In one embodiment, the promoter may be a combination of two or morecomponents such as, but not limited to, CMV and CBA. Each component mayhave a length of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,310, 320, 330, 340, 350, 360, 370, 380, 381, 382, 383, 384, 385, 386,387, 388, 389, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490,500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630,640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770,780, 790, 800 or more than 800. Each component may have a length between200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500,300-600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600,500-700, 500-800, 600-700, 600-800 or 700-800. As a non-limitingexample, the promoter is a combination of a 382 nucleotide CMV-enhancersequence and a 260 nucleotide CBA-promoter sequence.

In one embodiment, the vector genome comprises at least one element toenhance the target specificity and expression (See e.g., Powell et al.Viral Expression Cassette Elements to Enhance Transgene TargetSpecificity and Expression in Gene Therapy, 2015; the contents of whichare herein incorporated by reference in its entirety). Non-limitingexamples of elements to enhance the transgene target specificity andexpression include promoters, endogenous miRNAs, post-transcriptionalregulatory elements (PREs), polyadenylation (PolyA) signal sequences andupstream enhancers (USEs), CMV enhancers and introns.

In one embodiment, the vector genome comprises at least one element toenhance the target specificity and expression (See e.g., Powell et al.Viral Expression Cassette Elements to Enhance Transgene TargetSpecificity and Expression in Gene Therapy, 2015; the contents of whichare herein incorporated by reference in its entirety) such as promoters.

Promoters for which promote expression in most tissues include, but arenot limited to, human elongation factor 1α-subunit (EF1α),immediate-early cytomegalovirus (CMV), chicken β-actin (CBA) and itsderivative CAG, the β glucuronidase (GUSB), or ubiquitin C (UBC).Tissue-specific expression elements can be used to restrict expressionto certain cell types such as, but not limited to, nervous systempromoters which can be used to restrict expression to neurons,astrocytes, or oligodendrocytes. Non-limiting example of tissue-specificexpression elements for neurons include neuron-specific enolase (NSE),platelet-derived growth factor (PDGF), platelet-derived growth factorB-chain (PDGF-β), the synapsin (Syn), the methyl-CpG binding protein 2(MeCP2), CaMKII, mGluR2, NFL, NFH, nβ2, PPE, Enk and EAAT2 promoters. Anon-limiting example of a tissue-specific expression elements forastrocytes include the glial fibrillary acidic protein (GFAP) and EAAT2promoters. A non-limiting example of a tissue-specific expressionelement for oligodendrocytes include the myelin basic protein (MBP)promoter.

In one embodiment, the vector genome comprises a ubiquitous promoter.Non-limiting examples of ubiquitous promoters include CMV, CBA(including derivatives CAG, CBh, etc.), EF-1α, PGK, UBC, GUSB (hGBp),and UCOE (promoter of HNRPA2B1-CBX3). Yu et al. (Molecular Pain 2011,7:63; the contents of which are herein incorporated by reference in itsentirety) evaluated the expression of eGFP under the CAG, EFIα, PGK andUBC promoters in rat DRG cells and primary DRG cells using lentiviralvectors and found that UBC showed weaker expression than the other 3promoters and there was only 10-12% glia expression seen for allpromoters. Soderblom et al. (E. Neuro 2015; the contents of which areherein incorporated by reference in its entirety) the expression of eGFPin AAV8 with CMV and UBC promoters and AAV2 with the CMV promoter afterinjection in the motor cortex. Intranasal administration of a plasmidcontaining a UBC or EFIα promoter showed a sustained airway expressiongreater than the expression with the CMV promoter (See e.g., Gill etal., Gene Therapy 2001, Vol. 8, 1539-1546; the contents of which areherein incorporated by reference in its entirety). Husain et al. (GeneTherapy 2009; the contents of which are herein incorporated by referencein its entirety) evaluated a HβH construct with a hGUSB promoter, aHSV-1LAT promoter and a NSE promoter and found that the HβH constructshowed weaker expression than NSE in mice brain. Passini and Wolfe (J.Virol. 2001, 12382-12392, the contents of which are herein incorporatedby reference in its entirety) evaluated the long-term effects of the HβHvector following an intraventricular injection in neonatal mice andfound that there was sustained expression for at least 1 year. Lowexpression in all brain regions was found by Xu et al. (Gene Therapy2001, 8, 1323-1332; the contents of which are herein incorporated byreference in its entirety) when NF-L and NF-H promoters were used ascompared to the CMV-lacZ, CMV-luc, EF, GFAP, hENK, nAChR, PPE, PPE+wpre,NSE (0.3 kb), NSE (1.8 kb) and NSE (1.8 kb+wpre). Xu et al. found thatthe promoter activity in descending order was NSE (1.8 kb), EF, NSE (0.3kb), GFAP, CMV, hENK, PPE, NFL and NFH. NFL is a 650-nucleotide promoterand NFH is a 920-nucleotide promoter which are both absent in the liverbut NFH is abundant in the sensory proprioceptive neurons, brain andspinal cord and NFH is present in the heart. Scn8a is a 470-nucleotidepromoter which expresses throughout the DRG, spinal cord and brain withparticularly high expression seen in the hippocampal neurons andcerebellar Purkinje cells, cortex, thalmus and hypothalamus (See e.g.,Drews et al. 2007 and Raymond et al. 2004; the contents of each of whichare herein incorporated by reference in their entireties).

In one embodiment, the vector genome comprises an UBC promoter. The UBCpromoter may have a size of 300-350 nucleotides. As a non-limitingexample, the UBC promoter is 332 nucleotides.

In one embodiment, the vector genome comprises a GUSB promoter. The GUSBpromoter may have a size of 350-400 nucleotides. As a non-limitingexample, the GUSB promoter is 378 nucleotides. As a non-limitingexample, the construct may be AAV-promoter-CMV/globin intron-hFXN-RBG,where the AAV may be self-complementary and the AAV may be the DJserotype.

In one embodiment, the vector genome comprises a NFL promoter. The NFLpromoter may have a size of 600-700 nucleotides. As a non-limitingexample, the NFL promoter is 650 nucleotides. As a non-limiting example,the construct may be AAV-promoter-CMV/globin intron-hFXN-RBG, where theAAV may be self-complementary and the AAV may be the DJ serotype.

In one embodiment, the vector genome comprises a NFH promoter. The NFHpromoter may have a size of 900-950 nucleotides. As a non-limitingexample, the NFH promoter is 920 nucleotides. As a non-limiting example,the construct may be AAV-promoter-CMV/globin intron-hFXN-RBG, where theAAV may be self-complementary and the AAV may be the DJ serotype.

In one embodiment, the vector genome comprises a scn8a promoter. Thescn8a promoter may have a size of 450-500 nucleotides. As a non-limitingexample, the scn8a promoter is 470 nucleotides. As a non-limitingexample, the construct may be AAV-promoter-CMV/globin intron-hFXN-RBG,where the AAV may be self-complementary and the AAV may be the DJserotype.

In one embodiment, the vector genome comprises a FXN promoter.

In one embodiment, the vector genome comprises a PGK promoter.

In one embodiment, the vector genome comprises a CBA promoter.

In one embodiment, the vector genome comprises a CMV promoter.

In one embodiment, the vector genome comprises a liver or a skeletalmuscle promoter. Non-limiting examples of liver promoters include hAATand TBG. Non-limiting examples of skeletal muscle promoters includeDesmin, MCK and C5-12.

In one embodiment, the AAV vector comprises an enhancer element, apromoter and/or a 5′UTR intron. The enhancer may be, but is not limitedto, a CMV enhancer, the promoter may be, but is not limited to, a CMV,CBA, UBC, GUSB, NSE, Sunapsin, MeCP2, and GFAP promoter and the5′UTR/intron may be, but is not limited to, SV40, and CBA-MVM. As anon-limiting example, the enhancer, promoter and/or intron used incombination may be: (1) CMV enhancer, CMV promoter, SV40 5′UTR intron;(2) CMV enhancer, CBA promoter, SV 40 5′UTR intron; (3) CMV enhancer,CBA promoter, CBA-MVM 5′UTR intron; (4) UBC promoter; (5) GUSB promoter;(6) NSE promoter; (7) Synapsin promoter; (8) MeCP2 promoter and (9) GFAPpromoter.

In one embodiment, the AAV vector has an engineered promoter.

Introns

In one embodiment, the vector genome comprises at least one element toenhance the transgene target specificity and expression (See e.g.,Powell et al. Viral Expression Cassette Elements to Enhance TransgeneTarget Specificity and Expression in Gene Therapy, 2015; the contents ofwhich are herein incorporated by reference in its entirety) such as anintron. Non-limiting examples of introns include, MVM (67-97 bps), F. IXtruncated intron 1 (300 bps), β-globin SD/immunoglobulin heavy chainsplice acceptor (250 bps), adenovirus splice donor/immunoglobin spliceacceptor (500 bps), SV40 late splice donor/splice acceptor (19S/16S)(180 bps) and hybrid adenovirus splice donor/IgG splice acceptor (230bps).

In one embodiment, the intron may be 100-500 nucleotides in length. Theintron may have a length of 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 190, 200, 210,220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350,360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 or500. The promoter may have a length between 80-100, 80-120, 80-140,80-160, 80-180, 80-200, 80-250, 80-300, 80-350, 80-400, 80-450, 80-500,200-300, 200-400, 200-500, 300-400, 300-500, or 400-500.

In one embodiment, the AAV vector genome may comprise a promoter suchas, but not limited to, CMV or U6. As a non-limiting example, thepromoter for the AAV comprising the nucleic acid sequence for the siRNAmolecules of the present invention is a CMV promoter. As anothernon-limiting example, the promoter for the AAV comprising the nucleicacid sequence for the siRNA molecules of the present invention is a U6promoter.

In one embodiment, the AAV vector may comprise a CMV and a U6 promoter.

In one embodiment, the AAV vector may comprise a CBA promoter.

Introduction into Cells—Synthetic dsRNA

To ensure the chemical and biological stability of siRNA molecules(e.g., siRNA duplexes and dsRNA), it is important to deliver siRNAmolecules inside the target cells. In some embodiments, the cells mayinclude, but are not limited to, cells of mammalian origin, cells ofhuman origins, embryonic stem cells, induced pluripotent stem cells,neural stem cells, and neural progenitor cells.

Nucleic acids, including siRNA, carry a net negative charge on thesugar-phosphate backbone under normal physiological conditions. In orderto enter the cell, a siRNA molecule must come into contact with a lipidbilayer of the cell membrane, whose head groups are also negativelycharged.

The siRNA duplexes can be complexed with a carrier that allows them totraverse cell membranes such as package particles to facilitate cellularuptake of the siRNA. The package particles may include, but are notlimited to, liposomes, nanoparticles, cationic lipids, polyethyleniminederivatives, dendrimers, carbon nanotubes and the combination ofcarbon-made nanoparticles with dendrimers. Lipids may be cationic lipidsand/or neutral lipids. In addition to well established lipophiliccomplexes between siRNA molecules and cationic carriers, siRNA moleculescan be conjugated to a hydrophobic moiety, such as cholesterol (e.g.,U.S. Patent Publication No. 20110110937; the content of which is hereinincorporated by reference in its entirety). This delivery method holds apotential of improving in vitro cellular uptake and in vivopharmacological properties of siRNA molecules. The siRNA molecules ofthe present invention may also be conjugated to certain cationiccell-penetrating peptides (CPPs), such as MPG, transportan or penetratincovalently or non-covalently (e.g., U.S. Patent Publication No.20110086425; the content of which is herein incorporated by reference inits entirety).

Introduction into Cells—AAV Vectors

The siRNA molecules (e.g., siRNA duplexes) of the present invention maybe introduced into cells using any of a variety of approaches such as,but not limited to, viral vectors (e.g., AAV vectors). These viralvectors are engineered and optimized to facilitate the entry of siRNAmolecule into cells that are not readily amendable to transfection.Also, some synthetic viral vectors possess an ability to integrate theshRNA into the cell genome, thereby leading to stable siRNA expressionand long-term knockdown of a target gene. In this manner, viral vectorsare engineered as vehicles for specific delivery while lacking thedeleterious replication and/or integration features found in wild-typevirus.

In some embodiments, the siRNA molecules of the present invention areintroduced into a cell by contacting the cell with a compositioncomprising a lipophilic carrier and a vector, e.g., an AAV vector,comprising a nucleic acid sequence encoding the siRNA molecules of thepresent invention. In other embodiments, the siRNA molecule isintroduced into a cell by transfecting or infecting the cell with avector, e.g., an AAV vector, comprising nucleic acid sequences capableof producing the siRNA molecule when transcribed in the cell. In someembodiments, the siRNA molecule is introduced into a cell by injectinginto the cell a vector, e.g., an AAV vector, comprising a nucleic acidsequence capable of producing the siRNA molecule when transcribed in thecell.

In some embodiments, prior to transfection, a vector, e.g., an AAVvector, comprising a nucleic acid sequence encoding the siRNA moleculesof the present invention may be transfected into cells.

In other embodiments, the vectors, e.g., AAV vectors, comprising thenucleic acid sequence encoding the siRNA molecules of the presentinvention may be delivered into cells by electroporation (e.g., U.S.Patent Publication No. 20050014264; the content of which is hereinincorporated by reference in its entirety).

Other methods for introducing vectors, e.g., AAV vectors, comprising thenucleic acid sequence for the siRNA molecules described herein mayinclude photochemical internalization as described in U. S. Patentpublication No. 20120264807; the content of which is herein incorporatedby reference in its entirety.

In some embodiments, the formulations described herein may contain atleast one vector, e.g., AAV vectors, comprising the nucleic acidsequence encoding the siRNA molecules described herein. In oneembodiment, the siRNA molecules may target the SOD1 gene at one targetsite. In another embodiment, the formulation comprises a plurality ofvectors, e.g., AAV vectors, each vector comprising a nucleic acidsequence encoding a siRNA molecule targeting the SOD1 gene at adifferent target site. The SOD1 may be targeted at 2, 3, 4, 5 or morethan 5 sites.

In one embodiment, the vectors, e.g., AAV vectors, from any relevantspecies, such as, but not limited to, human, dog, mouse, rat or monkeymay be introduced into cells.

In one embodiment, the vectors, e.g., AAV vectors, may be introducedinto cells which are relevant to the disease to be treated. As anon-limiting example, the disease is ALS and the target cells are motorneurons and astrocytes.

In one embodiment, the vectors, e.g., AAV vectors, may be introducedinto cells which have a high level of endogenous expression of thetarget sequence.

In another embodiment, the vectors, e.g., AAV vectors, may be introducedinto cells which have a low level of endogenous expression of the targetsequence.

In one embodiment, the cells may be those which have a high efficiencyof AAV transduction.

Pharmaceutical Compositions and Formulation

In addition to the pharmaceutical compositions (vectors, e.g., AAVvectors, comprising a nucleic acid sequence encoding the siRNAmolecules), provided herein are pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to any other animal, e.g., to non-human animals, e.g.,non-human mammals. Modification of pharmaceutical compositions suitablefor administration to humans in order to render the compositionssuitable for administration to various animals is well understood, andthe ordinarily skilled veterinary pharmacologist can design and/orperform such modification with merely ordinary, if any, experimentation.Subjects to which administration of the pharmaceutical compositions iscontemplated include, but are not limited to, humans and/or otherprimates; mammals, including commercially relevant mammals such ascattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/orbirds, including commercially relevant birds such as poultry, chickens,ducks, geese, and/or turkeys.

In some embodiments, compositions are administered to humans, humanpatients, or subjects. For the purposes of the present disclosure, thephrase “active ingredient” generally refers either to the syntheticsiRNA duplexes, the vector, e.g., AAV vector, encoding the siRNAduplexes, or to the siRNA molecule delivered by a vector as describedherein.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, dividing, shaping and/or packaging the product into a desiredsingle- or multi-dose unit.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the invention will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered.

The vectors e.g., AAV vectors, comprising the nucleic acid sequenceencoding the siRNA molecules of the present invention can be formulatedusing one or more excipients to: (1) increase stability; (2) increasecell transfection or transduction; (3) permit the sustained or delayedrelease; or (4) alter the biodistribution (e.g., target the viral vectorto specific tissues or cell types such as brain and motor neurons).

Formulations of the present invention can include, without limitation,saline, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes,core-shell nanoparticles, peptides, proteins, cells transfected withviral vectors (e.g., for transplantation into a subject), nanoparticlemimics and combinations thereof. Further, the viral vectors of thepresent invention may be formulated using self-assembled nucleic acidnanoparticles.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofassociating the active ingredient with an excipient and/or one or moreother accessory ingredients.

A pharmaceutical composition in accordance with the present disclosuremay be prepared, packaged, and/or sold in bulk, as a single unit dose,and/or as a plurality of single unit doses. As used herein, a “unitdose” refers to a discrete amount of the pharmaceutical compositioncomprising a predetermined amount of the active ingredient. The amountof the active ingredient is generally equal to the dosage of the activeingredient which would be administered to a subject and/or a convenientfraction of such a dosage such as, for example, one-half or one-third ofsuch a dosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the present disclosure mayvary, depending upon the identity, size, and/or condition of the subjectbeing treated and further depending upon the route by which thecomposition is to be administered. For example, the composition maycomprise between 0.1% and 99% (w/w) of the active ingredient. By way ofexample, the composition may comprise between 0.1% and 100%, e.g.,between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w)active ingredient.

In some embodiments, a pharmaceutically acceptable excipient may be atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% pure. In some embodiments, an excipient is approved for use forhumans and for veterinary use. In some embodiments, an excipient may beapproved by United States Food and Drug Administration. In someembodiments, an excipient may be of pharmaceutical grade. In someembodiments, an excipient may meet the standards of the United StatesPharmacopoeia (USP), the European Pharmacopoeia (EP), the BritishPharmacopoeia, and/or the International Pharmacopoeia.

Excipients, which, as used herein, includes, but is not limited to, anyand all solvents, dispersion media, diluents, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, and the like, as suitedto the particular dosage form desired. Various excipients forformulating pharmaceutical compositions and techniques for preparing thecomposition are known in the art (see Remington: The Science andPractice of Pharmacy, 21^(st) Edition, A. R. Gennaro, Lippincott,Williams & Wilkins, Baltimore, Md., 2006; incorporated herein byreference in its entirety). The use of a conventional excipient mediummay be contemplated within the scope of the present disclosure, exceptinsofar as any conventional excipient medium may be incompatible with asubstance or its derivatives, such as by producing any undesirablebiological effect or otherwise interacting in a deleterious manner withany other component(s) of the pharmaceutical composition.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc.,and/or combinations thereof.

In some embodiments, the formulations may comprise at least one inactiveingredient. As used herein, the term “inactive ingredient” refers to oneor more inactive agents included in formulations. In some embodiments,all, none, or some of the inactive ingredients which may be used in theformulations of the present invention may be approved by the US Food andDrug Administration (FDA).

Formulations of vectors comprising the nucleic acid sequence for thesiRNA molecules of the present invention may include cations or anions.In one embodiment, the formulations include metal cations such as, butnot limited to, Zn2+, Ca2+, Cu2+, Mg+ and combinations thereof.

As used herein, “pharmaceutically acceptable salts” refers toderivatives of the disclosed compounds wherein the parent compound ismodified by converting an existing acid or base moiety to its salt form(e.g., by reacting the free base group with a suitable organic acid).Examples of pharmaceutically acceptable salts include, but are notlimited to, mineral or organic acid salts of basic residues such asamines; alkali or organic salts of acidic residues such as carboxylicacids; and the like. Representative acid addition salts include acetate,acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate,benzene sulfonic acid, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like, aswell as nontoxic ammonium, quaternary ammonium, and amine cations,including, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like. The pharmaceutically acceptablesalts of the present disclosure include the conventional non-toxic saltsof the parent compound formed, for example, from non-toxic inorganic ororganic acids. The pharmaceutically acceptable salts of the presentdisclosure can be synthesized from the parent compound which contains abasic or acidic moiety by conventional chemical methods. Generally, suchsalts can be prepared by reacting the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin water or in an organic solvent, or in a mixture of the two;generally, nonaqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are preferred. Lists of suitable salts arefound in Remington's Pharmaceutical Sciences, 17^(th) ed., MackPublishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts:Properties, Selection, and Use, P. H. Stahl and C. G. Wermuth (eds.),Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science,66, 1-19 (1977); the content of each of which is incorporated herein byreference in their entirety.

The term “pharmaceutically acceptable solvate,” as used herein, means acompound of the invention wherein molecules of a suitable solvent areincorporated in the crystal lattice. A suitable solvent isphysiologically tolerable at the dosage administered. For example,solvates may be prepared by crystallization, recrystallization, orprecipitation from a solution that includes organic solvents, water, ora mixture thereof. Examples of suitable solvents are ethanol, water (forexample, mono-, di-, and tri-hydrates), N-methylpyrrolidinone (NMP),dimethyl sulfoxide (DMSO), N,N′-dimethylformamide (DMF),N,N′-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU),1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile(ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone,benzyl benzoate, and the like. When water is the solvent, the solvate isreferred to as a “hydrate.”

According to the present invention, the vector, e.g., AAV vector,comprising the nucleic acid sequence for the siRNA molecules of thepresent invention may be formulated for CNS delivery. Agents that crossthe brain blood barrier may be used. For example, some cell penetratingpeptides that can target siRNA molecules to the brain blood barrierendothelium may be used to formulate the siRNA duplexes targeting theSOD1 gene (e.g., Mathupala, Expert Opin Ther Pat., 2009, 19, 137-140;the content of which is incorporated herein by reference in itsentirety).

Administration

The vector, e.g., AAV vector, comprising a nucleic acid sequenceencoding the siRNA molecules of the present invention may beadministered by any route which results in a therapeutically effectiveoutcome. These include, but are not limited to enteral (into theintestine), gastroenteral, epidural (into the dura matter), oral (by wayof the mouth), transdermal, peridural, intracerebral (into thecerebrum), intracerebroventricular (into the cerebral ventricles),epicutaneous (application onto the skin), intradermal, (into the skinitself), subcutaneous (under the skin), nasal administration (throughthe nose), intravenous (into a vein), intravenous bolus, intravenousdrip, intraarterial (into an artery), intramuscular (into a muscle),intracardiac (into the heart), intraosseous infusion (into the bonemarrow), intrathecal (into the spinal canal), intraperitoneal, (infusionor injection into the peritoneum), intravesical infusion, intravitreal,(through the eye), intracavernous injection (into a pathologic cavity)intracavitary (into the base of the penis), intravaginal administration,intrauterine, extra-amniotic administration, transdermal (diffusionthrough the intact skin for systemic distribution), transmucosal(diffusion through a mucous membrane), transvaginal, insufflation(snorting), sublingual, sublabial, enema, eye drops (onto theconjunctiva), in ear drops, auricular (in or by way of the ear), buccal(directed toward the cheek), conjunctival, cutaneous, dental (to a toothor teeth), electro-osmosis, endocervical, endosinusial, endotracheal,extracorporeal, hemodialysis, infiltration, interstitial,intra-abdominal, intra-amniotic, intra-articular, intrabiliary,intrabronchial, intrabursal, intracartilaginous (within a cartilage),intracaudal (within the cauda equine), intracisternal (within thecisterna magna cerebellomedularis), intracorneal (within the cornea),dental intracornal, intracoronary (within the coronary arteries),intracorporus cavernosum (within the dilatable spaces of the corporuscavernosa of the penis), intradiscal (within a disc), intraductal(within a duct of a gland), intraduodenal (within the duodenum),intradural (within or beneath the dura), intraepidermal (to theepidermis), intraesophageal (to the esophagus), intragastric (within thestomach), intragingival (within the gingivae), intraileal (within thedistal portion of the small intestine), intralesional (within orintroduced directly to a localized lesion), intraluminal (within a lumenof a tube), intralymphatic (within the lymph), intramedullary (withinthe marrow cavity of a bone), intrameningeal (within the meninges),intraocular (within the eye), intraovarian (within the ovary),intrapericardial (within the pericardium), intrapleural (within thepleura), intraprostatic (within the prostate gland), intrapulmonary(within the lungs or its bronchi), intrasinal (within the nasal orperiorbital sinuses), intraspinal (within the vertebral column),intrasynovial (within the synovial cavity of a joint), intratendinous(within a tendon), intratesticular (within the testicle), intrathecal(within the cerebrospinal fluid at any level of the cerebrospinal axis),intrathoracic (within the thorax), intratubular (within the tubules ofan organ), intratumor (within a tumor), intratympanic (within the aurusmedia), intravascular (within a vessel or vessels), intraventricular(within a ventricle), iontophoresis (by means of electric current whereions of soluble salts migrate into the tissues of the body), irrigation(to bathe or flush open wounds or body cavities), laryngeal (directlyupon the larynx), nasogastric (through the nose and into the stomach),occlusive dressing technique (topical route administration which is thencovered by a dressing which occludes the area), ophthalmic (to theexternal eye), oropharyngeal (directly to the mouth and pharynx),parenteral, percutaneous, periarticular, peridural, perineural,periodontal, rectal, respiratory (within the respiratory tract byinhaling orally or nasally for local or systemic effect), retrobulbar(behind the pons or behind the eyeball), soft tissue, subarachnoid,subconjunctival, submucosal, topical, transplacental (through or acrossthe placenta), transtracheal (through the wall of the trachea),transtympanic (across or through the tympanic cavity), ureteral (to theureter), urethral (to the urethra), vaginal, caudal block, diagnostic,nerve block, biliary perfusion, cardiac perfusion, photopheresis orspinal.

In specific embodiments, compositions of vector, e.g., AAV vector,comprising a nucleic acid sequence encoding the siRNA molecules of thepresent invention may be administered in a way which facilitates thevectors or siRNA molecule to enter the central nervous system andpenetrate into motor neurons.

In some embodiments, the vector, e.g., AAV vector, comprising a nucleicacid sequence encoding the siRNA molecules of the present invention maybe administered by muscular injection. Rizvanov et al. demonstrated forthe first time that siRNA molecules, targeting mutant human SOD1 mRNA,is taken up by the sciatic nerve, retrogradely transported to theperikarya of motor neurons, and inhibits mutant SOD1 mRNA in SOD1^(G93A)transgenic ALS mice (Rizvanov A A et al., Exp. Brain Res., 2009, 195(1),1-4; the content of which is incorporated herein by reference in itsentirety). Another study also demonstrated that muscle delivery of AAVexpressing small hairpin RNAs (shRNAs) against the mutant SOD1 gene, ledto significant mutant SOD1 knockdown in the muscle as well asinnervating motor neurons (Towne C et al., Mol Ther., 2011; 19(2):274-283; the content of which is incorporated herein by reference in itsentirety).

In some embodiments, AAV vectors that express siRNA duplexes of thepresent invention may be administered to a subject by peripheralinjections and/or intranasal delivery. It was disclosed in the art thatthe peripheral administration of AAV vectors for siRNA duplexes can betransported to the central nervous system, for example, to the motorneurons (e.g., U.S. Patent Publication Nos. 20100240739; and20100130594; the content of each of which is incorporated herein byreference in their entirety).

In other embodiments, compositions comprising at least one vector, e.g.,AAV vector, comprising a nucleic acid sequence encoding the siRNAmolecules of the present invention may be administered to a subject byintracranial delivery (See, e.g., U.S. Pat. No. 8,119,611; the contentof which is incorporated herein by reference in its entirety).

The vector, e.g., AAV vector, comprising a nucleic acid sequenceencoding the siRNA molecules of the present invention may beadministered in any suitable form, either as a liquid solution orsuspension, as a solid form suitable for liquid solution or suspensionin a liquid solution. The siRNA duplexes may be formulated with anyappropriate and pharmaceutically acceptable excipient.

The vector, e.g., AAV vector, comprising a nucleic acid sequenceencoding the siRNA molecules of the present invention may beadministered in a “therapeutically effective” amount, i.e., an amountthat is sufficient to alleviate and/or prevent at least one symptomassociated with the disease, or provide improvement in the condition ofthe subject.

In one embodiment, the vector, e.g., AAV vector, may be administered tothe CNS in a therapeutically effective amount to improve function and/orsurvival for a subject with ALS. As a non-limiting example, the vectormay be administered intrathecally.

In one embodiment, the vector, e.g., AAV vector, may be administered toa subject (e.g., to the CNS of a subject via intrathecal administration)in a therapeutically effective amount for the siRNA duplexes or dsRNA totarget the motor neurons and astrocytes in the spinal cord and/or brainsteam. As a non-limiting example, the siRNA duplexes or dsRNA may reducethe expression of SOD1 protein or mRNA. As another non-limiting example,the siRNA duplexes or dsRNA can suppress SOD1 and reduce SOD1 mediatedtoxicity. The reduction of SOD1 protein and/or mRNA as well as SOD1mediated toxicity may be accomplished with almost no enhancedinflammation.

In one embodiment, the vector, e.g., AAV vector, may be administered toa subject (e.g., to the CNS of a subject) in a therapeutically effectiveamount to slow the functional decline of a subject (e.g., determinedusing a known evaluation method such as the ALS functional rating scale(ALSFRS)) and/or prolong ventilator-independent survival of subjects(e.g., decreased mortality or need for ventilation support). As anon-limiting example, the vector may be administered intrathecally.

In one embodiment, the vector, e.g., AAV vector, may be administered tothe cisterna magna in a therapeutically effective amount to transducespinal cord motor neurons and/or astrocytes. As a non-limiting example,the vector may be administered intrathecally.

In one embodiment, the vector, e.g., AAV vector, may be administeredusing intrathecal infusion in a therapeutically effective amount totransduce spinal cord motor neurons and/or astrocytes. As a non-limitingexample, the vector may be administered intrathecally.

In one embodiment, the vector, e.g., AAV vector, comprising a modulatorypolynucleotide may be formulated. As a non-limiting example the baricityand/or osmolality of the formulation may be optimized to ensure optimaldrug distribution in the central nervous system or a region or componentof the central nervous system.

In one embodiment, the vector, e.g., AAV vector, comprising a modulatorypolynucleotide may be delivered to a subject via a single routeadministration.

In one embodiment, the vector, e.g., AAV vector, comprising a modulatorypolynucleotide may be delivered to a subject via a multi-site route ofadministration. A subject may be administered the vector, e.g., AAVvector, comprising a modulatory polynucleotide at 2, 3, 4, 5 or morethan 5 sites.

In one embodiment, a subject may be administered the vector, e.g., AAVvector, comprising a modulatory polynucleotide described herein using abolus infusion.

In one embodiment, a subject may be administered the vector, e.g., AAVvector, comprising a modulatory polynucleotide described herein usingsustained delivery over a period of minutes, hours, or days. Theinfusion rate may be changed depending on the subject, distribution,formulation, or another delivery parameter.

In one embodiment, the catheter may be located at more than one site inthe spine for multi-site delivery. The vector, e.g., AAV vector,comprising a modulatory polynucleotide may be delivered in a continuousand/or bolus infusion. Each site of delivery may be a different dosingregimen or the same dosing regimen may be used for each site ofdelivery. As a non-limiting example, the sites of delivery may be in thecervical and the lumbar region. As another non-limiting example, thesites of delivery may be in the cervical region. As another non-limitingexample, the sites of delivery may be in the lumbar region.

In one embodiment, a subject may be analyzed for spinal anatomy andpathology prior to delivery of the vector, e.g., AAV vector, comprisinga modulatory polynucleotide described herein. As a non-limiting example,a subject with scoliosis may have a different dosing regimen and/orcatheter location compared to a subject without scoliosis.

In one embodiment, the orientation of the spine of the subject duringdelivery of the vector, e.g., AAV vector, comprising a modulatorypolynucleotide may be vertical to the ground.

In another embodiment, the orientation of the spine of the subjectduring delivery of the vector, e.g., AAV vector, comprising a modulatorypolynucleotide may be horizontal to the ground.

In one embodiment, the spine of the subject may be at an angle ascompared to the ground during the delivery of the vector, e.g., AAVvector, comprising a modulatory polynucleotide. The angle of the spineof the subject as compared to the ground may be at least 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or 180 degrees.

In one embodiment, the delivery method and duration is chosen to providebroad transduction in the spinal cord. As a non-limiting example,intrathecal delivery is used to provide broad transduction along therostral-caudal length of the spinal cord. As another non-limitingexample, multi-site infusions provide a more uniform transduction alongthe rostral-caudal length of the spinal cord. As yet anothernon-limiting example, prolonged infusions provide a more uniformtransduction along the rostral-caudal length of the spinal cord.

Dosing

The pharmaceutical compositions of the present invention may beadministered to a subject using any amount effective for reducing,preventing and/or treating a SOD1 associated disorder (e.g., ALS). Theexact amount required will vary from subject to subject, depending onthe species, age, and general condition of the subject, the severity ofthe disease, the particular composition, its mode of administration, itsmode of activity, and the like.

The compositions of the present invention are typically formulated inunit dosage form for ease of administration and uniformity of dosage. Itwill be understood, however, that the total daily usage of thecompositions of the present invention may be decided by the attendingphysician within the scope of sound medical judgment. The specifictherapeutic effectiveness for any particular patient will depend upon avariety of factors including the disorder being treated and the severityof the disorder; the activity of the specific compound employed; thespecific composition employed; the age, body weight, general health, sexand diet of the patient; the time of administration, route ofadministration, and rate of excretion of the siRNA duplexes employed;the duration of the treatment; drugs used in combination or coincidentalwith the specific compound employed; and like factors well known in themedical arts.

In one embodiment, the age and sex of a subject may be used to determinethe dose of the compositions of the present invention. As a non-limitingexample, a subject who is older may receive a larger dose (e.g., 5-10%,10-20%, 15-30%, 20-50%, 25-50% or at least 1%, 2%, 3%, 4%, 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90% more) of thecomposition as compared to a younger subject. As another non-limitingexample, a subject who is younger may receive a larger dose (e.g.,5-10%, 10-20%, 15-30%, 20-50%, 25-50% or at least 1%, 2%, 3%, 4%, 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90% more) ofthe composition as compared to an older subject. As yet anothernon-limiting example, a subject who is female may receive a larger dose(e.g., 5-10%, 10-20%, 15-30%, 20-50%, 25-50% or at least 1%, 2%, 3%, 4%,5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90% more)of the composition as compared to a male subject. As yet anothernon-limiting example, a subject who is male may receive a larger dose(e.g., 5-10%, 10-20%, 15-30%, 20-50%, 25-50% or at least 1%, 2%, 3%, 4%,5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90% more)of the composition as compared to a female subject

In some specific embodiments, the doses of AAV vectors for deliveringsiRNA duplexes of the present invention may be adapted dependent on thedisease condition, the subject, and the treatment strategy.

In one embodiment, delivery of the compositions in accordance with thepresent invention to cells comprises a rate of delivery defined by[VG/hour=mL/hour*VG/mL] wherein VG is viral genomes, VG/mL iscomposition concentration, and mL/hour is rate of prolonged delivery.

In one embodiment, delivery of compositions in accordance with thepresent invention to cells may comprise a total concentration persubject between about 1×10⁶ VG and about 1×10¹⁶ VG. In some embodiments,delivery may comprise a composition concentration of about 1×10⁶, 2×10⁶,3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷,4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸,5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹,6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰,6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 2.1×10¹¹, 2.2×10¹¹,2.3×10¹¹, 2.4×10¹¹, 2.5×10¹¹, 2.6×10¹¹, 2.7×10¹¹, 2.8×10¹¹, 2.9×10¹¹,3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 7.1×10¹¹, 7.2×10¹¹, 7.3×10¹¹,7.4×10¹¹, 7.5×10¹¹, 7.6×10¹¹, 7.7×10¹¹, 7.8×10¹¹, 7.9×10¹¹, 8×10¹¹,9×10¹¹, 1×10¹², 1.1×10¹², 1.2×10¹², 1.3×10¹², 1.4×10¹², 1.5×10¹²,1.6×10¹², 1.7×10¹², 1.8×10¹², 1.9×10¹², 2×10¹², 3×10¹², 4×10¹²,4.1×10¹², 4.2×10¹², 4.3×10¹², 4.4×10¹², 4.5×10¹², 4.6×10¹², 4.7×10¹²,4.8×10¹², 4.9×10¹², 5×10¹², 6×10¹², 7×10¹², 8×10¹², 8.1×10¹², 8.2×10¹²,8.3×10¹², 8.4×10¹², 8.5×10¹², 8.6×10¹², 8.7×10¹², 8.8×10¹², 8.9×10¹²,9×10¹², 1×10¹³, 2×10¹³, 3×10¹³, 4×10¹³, 5×10¹³, 6×10¹³, 6.7×10¹³,7×10¹³, 8×10¹³, 9×10¹³, 1×10¹⁴, 2×10¹⁴, 3×10¹⁴, 4×10¹⁴, 5×10¹⁴, 6×10¹⁴,7×10¹⁴, 8×10¹⁴, 9×10¹⁴, 1×10¹⁵, 2×10¹⁵, 3×10¹⁵, 4×10¹⁵, 5×10¹⁵, 6×10¹⁵,7×10¹⁵, 8×10¹⁵, 9×10¹⁵, or 1×10¹⁶ VG/subject.

In one embodiment, delivery of compositions in accordance with thepresent invention to cells may comprise a total concentration persubject between about 1×10⁶ VG/kg and about 1×10¹⁶ VG/kg. In someembodiments, delivery may comprise a composition concentration of about1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷,2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸,3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹,4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰,4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹,2.1×10¹¹, 2.2×10¹¹, 2.3×10¹¹, 2.4×10¹¹, 2.5×10¹¹, 2.6×10¹¹, 2.7×10¹¹,2.8×10¹¹, 2.9×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 7.1×10¹¹,7.2×10¹¹, 7.3×10¹¹, 7.4×10¹¹, 7.5×10¹¹, 7.6×10¹¹, 7.7×10¹¹, 7.8×10¹¹,7.9×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 1.1×10¹², 1.2×10¹², 1.3×10¹²,1.4×10¹², 1.5×10¹², 1.6×10¹², 1.7×10¹², 1.8×10¹², 1.9×10¹², 2×10¹²,3×10¹², 4×10¹², 4.1×10¹², 4.2×10¹², 4.3×10¹², 4.4×10¹², 4.5×10¹²,4.6×10¹², 4.7×10¹², 4.8×10¹², 4.9×10¹², 5×10¹², 6×10¹², 7×10¹², 8×10¹²,8.1×10¹², 8.2×10¹², 8.3×10¹², 8.4×10¹², 8.5×10¹², 8.6×10¹², 8.7×10′²,8.8×10¹², 8.9×10¹², 9×10¹², 1×10¹³, 2×10¹³, 3×10¹³, 4×10¹³, 5×10¹³,6×10¹³, 6.7×10¹³, 7×10¹³, 8×10¹³, 9×10¹³, 1×10¹⁴, 2×10¹⁴, 3×10¹⁴,4×10¹⁴, 5×10¹⁴, 6×10¹⁴, 7×10¹⁴, 8×10¹⁴, 9×10¹⁴, 1×10¹⁵, 2×10¹⁵, 3×10¹⁵,4×10¹⁵, 5×10¹⁵, 6×10¹⁵, 7×10¹⁵, 8×10¹⁵, 9×10¹⁵, or 1×10¹⁶ VG/kg.

In one embodiment, about 10⁵ to 10⁶ viral genome (unit) may beadministered per dose.

In one embodiment, delivery of the compositions in accordance with thepresent invention to cells may comprise a total concentration betweenabout 1×10⁶ VG/mL and about 1×10¹⁶ VG/mL. In some embodiments, deliverymay comprise a composition concentration of about 1×10⁶, 2×10⁶, 3×10⁶,4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷,5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸,6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹,7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰,7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹,7×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 1.1×10¹², 1.2×10¹², 1.3×10¹², 1.4×10¹²,1.5×10¹², 1.6×10¹², 1.7×10¹², 1.8×10¹², 1.9×10¹², 2×10¹², 2.1×10¹²,2.2×10¹², 2.3×10¹², 2.4×10¹², 2.5×10¹², 2.6×10¹², 2.7×10¹², 2.8×10¹²,2.9×10¹², 3×10¹², 3.1×10¹², 3.2×10¹², 3.3×10¹², 3.4×10¹², 3.5×10¹²,3.6×10¹², 3.7×10¹², 3.8×10¹², 3.9×10¹², 4×10¹², 4.1×10¹², 4.2×10¹²,4.3×10¹², 4.4×10¹², 4.5×10¹², 4.6×10¹², 4.7×10¹², 4.8×10¹², 4.9×10¹²,5×10¹², 6×10¹², 7×10¹², 8×10¹², 9×10¹², 1×10¹³, 2×10¹³, 3×10¹³, 4×10¹³,5×10¹³, 6×10′³, 6.7×10¹³, 7×10¹³, 8×10′³, 9×10¹³, 1×10¹⁴, 2×10¹⁴,3×10′⁴, 4×10¹⁴, 5×10¹⁴, 6×10¹⁴, 7×10¹⁴, 8×10¹⁴, 9×10¹⁴, 1×10¹⁵, 2×10¹⁵,3×10¹⁵, 4×10¹⁵, 5×10¹⁵, 6×10¹⁵ 7×10¹⁵, 8×10¹⁵, 9×10¹⁵, or 1×10¹⁶VG/mL.

In certain embodiments, the desired siRNA duplex dosage may be deliveredusing multiple administrations (e.g., two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or moreadministrations). When multiple administrations are employed, splitdosing regimens such as those described herein may be used. As usedherein, a “split dose” is the division of single unit dose or totaldaily dose into two or more doses, e.g., two or more administrations ofthe single unit dose. As used herein, a “single unit dose” is a dose ofany modulatory polynucleotide therapeutic administered in one dose/atone time/single route/single point of contact, i.e., singleadministration event. As used herein, a “total daily dose” is an amountgiven or prescribed in 24-hour period. It may be administered as asingle unit dose. In one embodiment, the viral vectors comprising themodulatory polynucleotides of the present invention are administered toa subject in split doses. They may be formulated in buffer only or in aformulation described herein.

Methods of Treatment of ALS

Provided in the present invention are methods for introducing thevectors, e.g., AAV vectors, comprising a nucleic acid sequence encodingthe siRNA molecules of the present invention into cells, the methodcomprising introducing into said cells any of the vectors in an amountsufficient for degradation of target SOD1 mRNA to occur, therebyactivating target-specific RNAi in the cells. In some aspects, the cellsmay be stem cells, neurons such as motor neurons, muscle cells and glialcells such as astrocytes.

Disclosed in the present invention are methods for treating ALSassociated with abnormal SOD1 function in a subject in need oftreatment. The method optionally comprises administering to the subjecta therapeutically effective amount of a composition comprising at leastvectors, e.g., AAV vectors, comprising a nucleic acid sequence encodingthe siRNA molecules of the present invention. As a non-limiting example,the siRNA molecules can silence SOD1 gene expression, inhibit SOD1protein production, and reduce one or more symptoms of ALS in thesubject such that ALS is therapeutically treated.

In some embodiments, the composition comprising the vectors, e.g., AAVvectors, comprising a nucleic acid sequence encoding the siRNA moleculesof the present invention is administered to the central nervous systemof the subject. In other embodiments, the composition comprising thevectors, e.g., AAV vectors, comprising a nucleic acid sequence encodingthe siRNA molecules of the present invention is administered to themuscles of the subject

In particular, the vectors, e.g., AAV vectors, comprising a nucleic acidsequence encoding the siRNA molecules of the present invention may bedelivered into specific types of targeted cells, including motorneurons; glial cells including oligodendrocyte, astrocyte, andmicroglia; and/or other cells surrounding neurons such as T cells.Studies in human ALS patients and animal SOD1 ALS models implicate glialcells as playing an early role in the dysfunction and death of motorneurons. Normal SOD1 in the surrounding, protective glial cells canprevent the motor neurons from dying even though mutant SOD1 is presentin motor neurons (e.g., reviewed by Philips and Rothstein, Exp. Neurol.,2014, May 22. pii: S0014-4886(14)00157-5; the content of which isincorporated herein by reference in its entirety).

In some specific embodiments, the vectors, e.g., AAV vectors, comprisinga nucleic acid sequence encoding the siRNA molecules of the presentinvention may be used as a therapy for ALS.

In some embodiments, the present composition is administered as a solotherapeutics or combination therapeutics for the treatment of ALS.

The vectors, e.g., AAV vectors, encoding siRNA duplexes targeting theSOD1 gene may be used in combination with one or more other therapeuticagents. By “in combination with,” it is not intended to imply that theagents must be administered at the same time and/or formulated fordelivery together, although these methods of delivery are within thescope of the present disclosure. Compositions can be administeredconcurrently with, prior to, or subsequent to, one or more other desiredtherapeutics or medical procedures. In general, each agent will beadministered at a dose and/or on a time schedule determined for thatagent.

Therapeutic agents that may be used in combination with the vectors,e.g., AAV vectors, encoding the nucleic acid sequence for the siRNAmolecules of the present invention can be small molecule compounds whichare antioxidants, anti-inflammatory agents, anti-apoptosis agents,calcium regulators, antiglutamatergic agents, structural proteininhibitors, and compounds involved in metal ion regulation.

Compounds tested for treating ALS which may be used in combination withthe vectors described herein include, but are not limited to,antiglutamatergic agents: Riluzole, Topiramate, Talampanel, Lamotrigine,Dextromethorphan, Gabapentin and AMPA antagonist; Anti-apoptosis agents:Minocycline, Sodium phenylbutyrate and Arimoclomol; Anti-inflammatoryagent: ganglioside, Celecoxib, Cyclosporine, Azathioprine,Cyclophosphamide, Plasmaphoresis, Glatiramer acetate and thalidomide;Ceftriaxone (Berry et al., Plos One, 2013, 8(4)); Beat-lactamantibiotics; Pramipexole (a dopamine agonist) (Wang et al., AmyotrophicLateral Scler., 2008, 9(1), 50-58); Nimesulide in U.S. PatentPublication No. 20060074991; Diazoxide disclosed in U.S. PatentPublication No. 20130143873); pyrazolone derivatives disclosed in USPatent Publication No. 20080161378; free radical scavengers that inhibitoxidative stress-induced cell death, such as bromocriptine (US. PatentPublication No. 20110105517); phenyl carbamate compounds discussed inPCT Patent Publication No. 2013100571; neuroprotective compoundsdisclosed in U.S. Pat. Nos. 6,933,310 and 8,399,514 and US PatentPublication Nos. 20110237907 and 20140038927; and glycopeptides taughtin U.S. Patent Publication No. 20070185012; the content of each of whichis incorporated herein by reference in their entirety.

Therapeutic agents that may be used in combination therapy with thevectors, e.g., AAV vectors, encoding the nucleic acid sequence for thesiRNA molecules of the present invention may be hormones or variantsthat can protect neuronal loss, such as adrenocorticotropic hormone(ACTH) or fragments thereof (e.g., U.S. Patent Publication No.20130259875); Estrogen (e.g., U.S. Pat. Nos. 6,334,998 and 6,592,845);the content of each of which is incorporated herein by reference intheir entirety.

Neurotrophic factors may be used in combination therapy with thevectors, e.g., AAV vectors, encoding the nucleic acid sequence for thesiRNA molecules of the present invention for treating ALS. Generally, aneurotrophic factor is defined as a substance that promotes survival,growth, differentiation, proliferation and/or maturation of a neuron, orstimulates increased activity of a neuron. In some embodiments, thepresent methods further comprise delivery of one or more trophic factorsinto the subject in need of treatment. Trophic factors may include, butare not limited to, IGF-I, GDNF, BDNF, CTNF, VEGF, Colivelin,Xaliproden, Thyrotrophin-releasing hormone and ADNF, and variantsthereof.

In one aspect, the vector, e.g., AAV vector, encoding the nucleic acidsequence for the at least one siRNA duplex targeting the SOD1 gene maybe co-administered with AAV vectors expressing neurotrophic factors suchas AAV-IGF-I (Vincent et al., Neuromolecular medicine, 2004, 6, 79-85;the content of which is incorporated herein by reference in itsentirety) and AAV-GDNF (Wang et al., J Neurosci., 2002, 22, 6920-6928;the content of which is incorporated herein by reference in itsentirety).

In some embodiments, the composition of the present invention fortreating ALS is administered to the subject in need intravenously,intramuscularly, subcutaneously, intraperitoneally, intrathecally and/orintraventricularly, allowing the siRNA molecules or vectors comprisingthe siRNA molecules to pass through one or both the blood-brain barrierand the blood spinal cord barrier. In some aspects, the method includesadministering (e.g., intraventricularly administering and/orintrathecally administering) directly to the central nervous system(CNS) of a subject (using, e.g., an infusion pump and/or a deliveryscaffold) a therapeutically effective amount of a composition comprisingvectors, e.g., AAV vectors, encoding the nucleic acid sequence for thesiRNA molecules of the present invention. The vectors may be used tosilence or suppress SOD1 gene expression, and/or reducing one or moresymptoms of ALS in the subject such that ALS is therapeutically treated.

In certain aspects, the symptoms of ALS include, but are not limited to,motor neuron degeneration, muscle weakness, muscle atrophy, thestiffness of muscle, difficulty in breathing, slurred speech,fasciculation development, frontotemporal dementia and/or prematuredeath are improved in the subject treated. In other aspects, thecomposition of the present invention is applied to one or both of thebrain and the spinal cord. In other aspects, one or both of musclecoordination and muscle function are improved. In other aspects, thesurvival of the subject is prolonged.

In one embodiment, administration of the vectors, e.g., AAV vectorsencoding a siRNA of the invention, to a subject may lower mutant SOD1 inthe CNS of a subject. In another embodiment, administration of thevectors, e.g., AAV vectors, to a subject may lower wild-type SOD1 in theCNS of a subject. In yet another embodiment, administration of thevectors, e.g., AAV vectors, to a subject may lower both mutant SOD1 andwild-type SOD1 in the CNS of a subject. The mutant and/or wild-type SOD1may be lowered by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%,20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%,30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%,40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%,60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%,80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100% in the CNS, a regionof the CNS, or a specific cell of the CNS of a subject. As anon-limiting example, the vectors, e.g., AAV vectors may lower theexpression of wild-type SOD1 by at least 50% in the motor neurons (e.g.,ventral horn motor neurons) and/or astrocytes. As another non-limitingexample, the vectors, e.g., AAV vectors may lower the expression ofmutant SOD1 by at least 50% in the motor neurons (e.g., ventral hornmotor neurons) and/or astrocytes. As yet another non-limiting example,the vectors, e.g., AAV vectors may lower the expression of wild-typeSOD1 and mutant SOD1 by at least 50% in the motor neurons (e.g., ventralhorn motor neurons) and/or astrocytes.

In one embodiment, administration of the vectors, e.g., AAV vectors, toa subject will reduce the expression of mutant and/or wild-type SOD1 inthe spinal cord and the reduction of expression of the mutant and/orwild-type SOD1 will reduce the effects of ALS in a subject.

In one embodiment, the vectors, e.g., AAV vectors, may be administeredto a subject who is in the early stages of ALS. Early-stage symptomsinclude, but are not limited to, muscles which are weak and soft orstiff, tight and spastic, cramping and twitching (fasciculations) ofmuscles, loss of muscle bulk (atrophy), fatigue, poor balance, slurredwords, weak grip, and/or tripping when walking. The symptoms may belimited to a single body region or a mild symptom may affect more thanone region. As a non-limiting example, administration of the vectors,e.g., AAV vectors, may reduce the severity and/or occurrence of thesymptoms of ALS.

In one embodiment, the vectors, e.g., AAV vectors, may be administeredto a subject who is in the middle stages of ALS. The middle stage of ALSincludes, but is not limited to, more widespread muscle symptoms ascompared to the early stage, some muscles are paralyzed while others areweakened or unaffected, continued muscle twitchings (fasciculations),unused muscles may cause contractures where the joints become rigid,painful and sometimes deformed, weakness in swallowing muscles may causechoking and greater difficulty eating and managing saliva, weakness inbreathing muscles can cause respiratory insufficiency which can beprominent when lying down, and/or a subject may have bouts ofuncontrolled and inappropriate laughing or crying (pseudobulbar affect).As a non-limiting example, administration of the vectors, e.g., AAVvectors, may reduce the severity and/or occurrence of the symptoms ofALS.

In one embodiment, the vectors, e.g., AAV vectors, may be administeredto a subject who is in the late stages of ALS. The late stage of ALSincludes, but is not limited to, voluntary muscles which are mostlyparalyzed, the muscles that help move air in and out of the lungs areseverely compromised, mobility is extremely limited, poor respirationmay cause fatigue, fuzzy thinking, headaches and susceptibility toinfection or diseases (e.g., pneumonia), speech is difficult and eatingor drinking by mouth may not be possible.

In one embodiment, the vectors, e.g., AAV vectors, may be used to treata subject with ALS who has a C9orf72 mutation.

In one embodiment, the vectors, e.g., AAV vectors, may be used to treata subject with ALS who has TDP-43 mutations.

In one embodiment, the vectors, e.g., AAV vectors, may be used to treata subject with ALS who has FUS mutations.

DEFINITIONS

Unless stated otherwise, the following terms and phrases have themeanings described below. The definitions are not meant to be limitingin nature and serve to provide a clearer understanding of certainaspects of the present invention.

As used herein, the term “nucleic acid”, “polynucleotide” and‘oligonucleotide” refer to any nucleic acid polymers composed of eitherpolydeoxyribonucleotides (containing 2-deoxy-D-ribose), orpolyribonucleotides (containing D-ribose), or any other type ofpolynucleotide which is an N glycoside of a purine or pyrimidine base,or modified purine or pyrimidine bases. There is no intended distinctionin length between the term “nucleic acid”, “polynucleotide” and“oligonucleotide”, and these terms will be used interchangeably. Theseterms refer only to the primary structure of the molecule. Thus, theseterms include double- and single-stranded DNA, as well as double- andsingle stranded RNA.

As used herein, the term “RNA” or “RNA molecule” or “ribonucleic acidmolecule” refers to a polymer of ribonucleotides; the term “DNA” or “DNAmolecule” or “deoxyribonucleic acid molecule” refers to a polymer ofdeoxyribonucleotides. DNA and RNA can be synthesized naturally, e.g., byDNA replication and transcription of DNA, respectively; or be chemicallysynthesized. DNA and RNA can be single-stranded (i.e., ssRNA or ssDNA,respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA anddsDNA, respectively). The term “mRNA” or “messenger RNA”, as usedherein, refers to a single stranded RNA that encodes the amino acidsequence of one or more polypeptide chains.

As used herein, the term “RNA interfering” or “RNAi” refers to asequence specific regulatory mechanism mediated by RNA molecules whichresults in the inhibition or interfering or “silencing” of theexpression of a corresponding protein-coding gene. RNAi has beenobserved in many types of organisms, including plants, animals andfungi. RNAi occurs in cells naturally to remove foreign RNAs (e.g.,viral RNAs). Natural RNAi proceeds via fragments cleaved from free dsRNAwhich direct the degradative mechanism to other similar RNA sequences.RNAi is controlled by the RNA-induced silencing complex (RISC) and isinitiated by short/small dsRNA molecules in cell cytoplasm, where theyinteract with the catalytic RISC component argonaute. The dsRNAmolecules can be introduced into cells exogenously. Exogenous dsRNAinitiates RNAi by activating the ribonuclease protein Dicer, which bindsand cleaves dsRNAs to produce double-stranded fragments of 21-25 basepairs with a few unpaired overhang bases on each end. These short doublestranded fragments are called small interfering RNAs (siRNAs).

As used herein, the terms “short interfering RNA,” “small interferingRNA” or “siRNA” refer to an RNA molecule (or RNA analog) comprisingbetween about 5-60 nucleotides (or nucleotide analogs) which is capableof directing or mediating RNAi. Preferably, a siRNA molecule comprisesbetween about 15-30 nucleotides or nucleotide analogs, such as betweenabout 16-25 nucleotides (or nucleotide analogs), between about 18-23nucleotides (or nucleotide analogs), between about 19-22 nucleotides (ornucleotide analogs) (e.g., 19, 20, 21 or 22 nucleotides or nucleotideanalogs), between about 19-25 nucleotides (or nucleotide analogs), andbetween about 19-24 nucleotides (or nucleotide analogs). The term“short” siRNA refers to a siRNA comprising 5-23 nucleotides, preferably21 nucleotides (or nucleotide analogs), for example, 19, 20, 21 or 22nucleotides. The term “long” siRNA refers to a siRNA comprising 24-60nucleotides, preferably about 24-25 nucleotides, for example, 23, 24, 25or 26 nucleotides. Short siRNAs may, in some instances, include fewerthan 19 nucleotides, e.g., 16, 17 or 18 nucleotides, or as few as 5nucleotides, provided that the shorter siRNA retains the ability tomediate RNAi. Likewise, long siRNAs may, in some instances, include morethan 26 nucleotides, e.g., 27, 28, 29, 30, 35, 40, 45, 50, 55, or even60 nucleotides, provided that the longer siRNA retains the ability tomediate RNAi or translational repression absent further processing,e.g., enzymatic processing, to a short siRNA. siRNAs can be singlestranded RNA molecules (ss-siRNAs) or double stranded RNA molecules(ds-siRNAs) comprising a sense strand and an antisense strand whichhybridized to form a duplex structure called siRNA duplex.

As used herein, the term “the antisense strand” or “the first strand” or“the guide strand” of a siRNA molecule refers to a strand that issubstantially complementary to a section of about 10-50 nucleotides,e.g., about 15-30, 16-25, 18-23 or 19-22 nucleotides of the mRNA of thegene targeted for silencing. The antisense strand or first strand hassequence sufficiently complementary to the desired target mRNA sequenceto direct target-specific silencing, e.g., complementarity sufficient totrigger the destruction of the desired target mRNA by the RNAi machineryor process.

As used herein, the term “the sense strand” or “the second strand” or“the passenger strand” of a siRNA molecule refers to a strand that iscomplementary to the antisense strand or first strand. The antisense andsense strands of a siRNA molecule are hybridized to form a duplexstructure. As used herein, a “siRNA duplex” includes a siRNA strandhaving sufficient complementarity to a section of about 10-50nucleotides of the mRNA of the gene targeted for silencing and a siRNAstrand having sufficient complementarity to form a duplex with the siRNAstrand.

As used herein, the term “complementary” refers to the ability ofpolynucleotides to form base pairs with one another. Base pairs aretypically formed by hydrogen bonds between nucleotide units inantiparallel polynucleotide strands. Complementary polynucleotidestrands can form base pair in the Watson-Crick manner (e.g., A to T, Ato U, C to G), or in any other manner that allows for the formation ofduplexes. As persons skilled in the art are aware, when using RNA asopposed to DNA, uracil rather than thymine is the base that isconsidered to be complementary to adenosine. However, when a U isdenoted in the context of the present invention, the ability tosubstitute a T is implied, unless otherwise stated. Perfectcomplementarity or 100% complementarity refers to the situation in whicheach nucleotide unit of one polynucleotide strand can form hydrogen bondwith a nucleotide unit of a second polynucleotide strand. Less thanperfect complementarity refers to the situation in which some, but notall, nucleotide units of two strands can form hydrogen bond with eachother. For example, for two 20-mers, if only two base pairs on eachstrand can form hydrogen bond with each other, the polynucleotidestrands exhibit 10% complementarity. In the same example, if 18 basepairs on each strand can form hydrogen bonds with each other, thepolynucleotide strands exhibit 90% complementarity.

As used herein, the term “substantially complementary” means that thesiRNA has a sequence (e.g., in the antisense strand) which is sufficientto bind the desired target mRNA, and to trigger the RNA silencing of thetarget mRNA.

As used herein, “targeting” means the process of design and selection ofnucleic acid sequence that will hybridize to a target nucleic acid andinduce a desired effect.

The term “gene expression” refers to the process by which a nucleic acidsequence undergoes successful transcription and in most instancestranslation to produce a protein or peptide. For clarity, when referenceis made to measurement of “gene expression”, this should be understoodto mean that measurements may be of the nucleic acid product oftranscription, e.g., RNA or mRNA or of the amino acid product oftranslation, e.g., polypeptides or peptides. Methods of measuring theamount or levels of RNA, mRNA, polypeptides, and peptides are well knownin the art.

As used herein, the term “mutation” refers to any changing of thestructure of a gene, resulting in a variant (also called “mutant”) formthat may be transmitted to subsequent generations. Mutations in a genemay be caused by the alternation of single base in DNA, or the deletion,insertion, or rearrangement of larger sections of genes or chromosomes.

As used herein, the term “vector” means any molecule or moiety whichtransports, transduces or otherwise acts as a carrier of a heterologousmolecule such as the siRNA molecule of the invention. A “viral vector”is a vector which comprises one or more polynucleotide regions encodingor comprising a molecule of interest, e.g., a transgene, apolynucleotide encoding a polypeptide or multi-polypeptide or amodulatory nucleic acid such as small interfering RNA (siRNA). Viralvectors are commonly used to deliver genetic materials into cells. Viralvectors are often modified for specific applications. Types of viralvectors include retroviral vectors, lentiviral vectors, adenoviralvectors and adeno-associated viral vectors.

The term “adeno-associated virus” or “AAV” or “AAV vector” as usedherein refers to any vector which comprises or derives from componentsof an adeno-associated vector and is suitable to infect mammalian cells,preferably human cells. The term AAV vector typically designates an AAVtype viral particle or virion comprising a nucleic acid moleculeencoding a siRNA duplex. The AAV vector may be derived from variousserotypes, including combinations of serotypes (i.e., “pseudotyped” AAV)or from various genomes (e.g., single stranded or self-complementary).In addition, the AAV vector may be replication defective and/ortargeted.

As used herein, the phrase “inhibit expression of a gene” means to causea reduction in the amount of an expression product of the gene. Theexpression product can be a RNA molecule transcribed from the gene(e.g., an mRNA) or a polypeptide translated from an mRNA transcribedfrom the gene. Typically a reduction in the level of an mRNA results ina reduction in the level of a polypeptide translated therefrom. Thelevel of expression may be determined using standard techniques formeasuring mRNA or protein.

As used herein, the term “in vitro” refers to events that occur in anartificial environment, e.g., in a test tube or reaction vessel, in cellculture, in a Petri dish, etc., rather than within an organism (e.g.,animal, plant, or microbe).

As used herein, the term “in vivo” refers to events that occur within anorganism (e.g., animal, plant, or microbe or cell or tissue thereof).

As used herein, the term “modified” refers to a changed state orstructure of a molecule of the invention. Molecules may be modified inmany ways including chemically, structurally, and functionally.

As used herein, the term “synthetic” means produced, prepared, and/ormanufactured by the hand of man. Synthesis of polynucleotides orpolypeptides or other molecules of the present invention may be chemicalor enzymatic.

As used herein, the term “transfection” refers to methods to introduceexogenous nucleic acids into a cell. Methods of transfection include,but are not limited to, chemical methods, physical treatments andcationic lipids or mixtures. The list of agents that can be transfectedinto a cell is large and includes, but is not limited to, siRNA, senseand/or anti-sense sequences, DNA encoding one or more genes andorganized into an expression plasmid, proteins, protein fragments, andmore.

As used herein, “off target” refers to any unintended effect on any oneor more target, gene, or cellular transcript.

As used herein, the phrase “pharmaceutically acceptable” is employedherein to refer to those compounds, materials, compositions, and/ordosage forms which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of human beings and animalswithout excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio.

As used herein, the term “effective amount” of an agent is that amountsufficient to effect beneficial or desired results, for example,clinical results, and, as such, an “effective amount” depends upon thecontext in which it is being applied. For example, in the context ofadministering an agent that treats ALS, an effective amount of an agentis, for example, an amount sufficient to achieve treatment, as definedherein, of ALS, as compared to the response obtained withoutadministration of the agent.

As used herein, the term “therapeutically effective amount” means anamount of an agent to be delivered (e.g., nucleic acid, drug,therapeutic agent, diagnostic agent, prophylactic agent, etc.) that issufficient, when administered to a subject suffering from or susceptibleto an infection, disease, disorder, and/or condition, to treat, improvesymptoms of, diagnose, prevent, and/or delay the onset of the infection,disease, disorder, and/or condition.

As used herein, the term “subject” or “patient” refers to any organismto which a composition in accordance with the invention may beadministered, e.g., for experimental, diagnostic, prophylactic, and/ortherapeutic purposes. Typical subjects include animals (e.g., mammalssuch as mice, rats, rabbits, non-human primates such as chimpanzees andother apes and monkey species, and humans) and/or plants.

As used herein, the term “preventing” or “prevention” refers to delayingor forestalling the onset, development or progression of a condition ordisease for a period of time, including weeks, months, or years.

The term “treatment” or “treating,” as used herein, refers to theapplication of one or more specific procedures used for the cure oramelioration of a disease. In certain embodiments, the specificprocedure is the administration of one or more pharmaceutical agents. Inthe context of the present invention, the specific procedure is theadministration of one or more siRNA duplexes or encoded dsRNA targetingSOD1 gene.

As used herein, the term “amelioration” or “ameliorating” refers to alessening of severity of at least one indicator of a condition ordisease. For example, in the context of neurodegeneration disorder,amelioration includes the reduction of neuron loss.

As used herein, the term “administering” refers to providing apharmaceutical agent or composition to a subject.

As used herein, the term “neurodegeneration” refers to a pathologicstate which results in neural cell death. A large number of neurologicaldisorders share neurodegeneration as a common pathological state. Forexample, Alzheimer's disease, Parkinson's disease, Huntington's disease,and amyotrophic lateral sclerosis (ALS) all cause chronicneurodegeneration, which is characterized by a slow, progressive neuralcell death over a period of several years, whereas acuteneurodegeneration is characterized by a sudden onset of neural celldeath as a result of ischemia, such as stroke, or trauma, such astraumatic brain injury, or as a result of axonal transection bydemyelination or trauma caused, for example, by spinal cord injury ormultiple sclerosis. In some neurological disorders, mainly one type ofneuron cells are degenerative, for example, motor neuron degeneration inALS.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the invention described herein. The scopeof the present invention is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or the entiregroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the term “consistingof” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anyantibiotic, therapeutic or active ingredient; any method of production;any method of use; etc.) can be excluded from any one or more claims,for any reason, whether or not related to the existence of prior art.

It is to be understood that the words which have been used are words ofdescription rather than limitation, and that changes may be made withinthe purview of the appended claims without departing from the true scopeand spirit of the invention in its broader aspects.

While the present invention has been described at some length and withsome particularity with respect to the several described embodiments, itis not intended that it should be limited to any such particulars orembodiments or any particular embodiment, but it is to be construed withreferences to the appended claims so as to provide the broadest possibleinterpretation of such claims in view of the prior art and, therefore,to effectively encompass the intended scope of the invention.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, section headings, the materials, methods, andexamples are illustrative only and not intended to be limiting.

EXAMPLES Example 1. SOD1 siRNA Design and Synthesis

SOD1 siRNA Design

siRNA design was carried out to identify siRNAs targeting human SOD1gene. The design used the SOD1 transcripts for human ((Genebank accessNO. NM_000454.4 (SEQ ID NO: 1)), cynomolgus ((Genebank access NO.XM_005548833.1) from the NCBI Refseq collection (release 63) (SEQ ID NO:2)) and rhesus (SOD1 transcript ENSMMUT00000002415 (SEQ ID NO: 3) fromthe Ensembl project (release 75)) as described in Table 2.

TABLE 2 SOD1 gene sequences SEQ ID SOD1 transcripts Access No. NO. HumanSOD1 cDNA (981 bp) NM_ 000454.4 1 cynomolgus SOD1 cDNA (465 bp)XM_005548833.1 2 rhesus SOD1 cDNA (464 bp) ENSMMUT00000002415 3

The siRNA duplexes were designed to have 100% identity to the human SOD1transcript for positions 2-18 of the antisense strand, and partial or100% identity to the non-human primate SOD1 transcript for positions2-18 of the antisense strand. In all siRNA duplexes, position 1 of theantisense strand was engineered to a U and position 19 of the sensestrand was engineered to a C, in order to unpair the duplex at thisposition.

SOD1 siRNA Sequence Selection

Based on predicted selectivity of the antisense strand for human,cynomolgus and rhesus SOD1 genes, and lack of match of the seed sequenceat positions 2-7 of the antisense strand to human sequences inmiRBase20.0, a total of 169 antisense and 169 sense human SOD1 derivedoligonucleotides were synthesized and formed into duplexes (Table 3).The siRNA duplexes were then tested for in vitro inhibitory activity onendogenous SOD1 gene expression (SOD1 mRNA levels).

TABLE 3 Sense and antisense strand sequences of human SOD1 dsRNA siRNASEQ duplex SS sense strand sequence ID AS antisense strand sequence SEQStart ID ID (5′-3′) NO ID (5′-3′) ID NO 26 D-2741 7414 CGGAGGUCUGGCCUA 47415 UUUAUAGGCCAGACCUCC 173 UAACdTdT GdTdT 27 D-2742 7416GGAGGUCUGGCCUAU 5 7417 UUUUAUAGGCCAGACCUC 174 AAACdTdT CdTdT 28 D-27437418 GAGGUCUGGCCUAUA 6 7419 UCUUUAUAGGCCAGACCU 175 AAGCdTdT CdTdT 29D-2744 7420 AGGUCUGGCCUAUAA 7 7421 UACUUUAUAGGCCAGACC 176 AGUCdTdT UdTdT30 D-2745 7422 GGUCUGGCCUAUAAA 8 7423 UUACUUUAUAGGCCAGAC 177 GUACdTdTCdTdT 32 D-2746 7424 UCUGGCCUAUAAAGU 9 7425 UACUACUUUAUAGGCCAG 178AGUCdTdT AdTdT 33 D-2747 7426 CUGGCCUAUAAAGUA 10 7427 UGACUACUUUAUAGGCCA179 GUCCdTdT GdTdT 34 D-2748 7428 UGGCCUAUAAAGUAG 11 7429UCGACUACUUUAUAGGCC 180 UCGCdTdT AdTdT 35 D-2749 7430 GGCCUAUAAAGUAGU 127431 UGCGACUACUUUAUAGGC 181 CGCCdTdT CdTdT 36 D-2750 7432GCCUAUAAAGUAGUC 13 7433 UCGCGACUACUUUAUAGG 182 GCGCdTdT CdTdT 37 D-27517434 CCUAUAAAGUAGUCG 14 7435 UCCGCGACUACUUUAUAG 183 CGGCdTdT GdTdT 74D-2752 7436 GUCGUAGUCUCCUGC 15 7437 UGCUGCAGGAGACUACGA 184 AGCCdTdTCdTdT 76 D-2753 7438 CGUAGUCUCCUGCAG 16 7439 UACGCUGCAGGAGACUAC 185CGUCdTdT GdTdT 77 D-2754 7440 GUAGUCUCCUGCAGC 17 7441 UGACGCUGCAGGAGACUA186 GUCCdTdT CdTdT 78 D-2755 7442 UAGUCUCCUGCAGCG 18 7443UAGACGCUGCAGGAGACU 187 UCUCdTdT AdTdT 149 D-2756 7444 AUGGCGACGAAGGCC 197445 UCACGGCCUUCGUCGCCA 188 GUGCdTdT UdTdT 153 D-2757 7446CGACGAAGGCCGUGU 20 7447 UCGCACACGGCCUUCGUC 189 GCGCdTdT GdTdT 157 D-27587448 GAAGGCCGUGUGCGU 21 7449 UAGCACGCACACGGCCUU 190 GCUCdTdT CdTdT 160D-2759 7450 GGCCGUGUGCGUGCU 22 7451 UUUCAGCACGCACACGGC 191 GAACdTdTCdTdT 177 D-2760 7452 AGGGCGACGGCCCAG 23 7453 UGCACUGGGCCGUCGCCC 192UGCCdTdT UdTdT 192 D-2761 7454 UGCAGGGCAUCAUCA 24 7455UAAUUGAUGAUGCCCUGC 193 AUUCdTdT AdTdT 193 D-2762 7456 GCAGGGCAUCAUCAA 257457 UAAAUUGAUGAUGCCCUG 194 UUUCdTdT CdTdT 195 D-2763 7458AGGGCAUCAUCAAUU 26 7459 UCGAAAUUGAUGAUGCCC 195 UCGCdTdT UdTdT 196 D-27647460 GGGCAUCAUCAAUUU 27 7461 UUCGAAAUUGAUGAUGCC 196 CGACdTdT CdTdT 197D-2765 7462 GGCAUCAUCAAUUUC 28 7463 UCUCGAAAUUGAUGAUGC 197 GAGCdTdTCdTdT 198 D-2766 7464 GCAUCAUCAAUUUCG 29 7465 UGCUCGAAAUUGAUGAUG 198AGCCdTdT CdTdT 199 D-2767 7466 CAUCAUCAAUUUCGA 30 7467UUGCUCGAAAUUGAUGAU 199 GCACdTdT GdTdT 206 D-2768 7468 AAUUUCGAGCAGAAG 317469 UUUCCUUCUGCUCGAAAU 200 GAACdTdT UdTdT 209 D-2769 7470UUCGAGCAGAAGGAA 32 7471 UACUUUCCUUCUGCUCGA 201 AGUCdTdT AdTdT 210 D-27707472 UCGAGCAGAAGGAAA 33 7473 UUACUUUCCUUCUGCUCG 202 GUACdTdT AdTdT 239D-2771 7474 AAGGUGUGGGGAAGC 34 7475 UAAUGCUUCCCCACACCU 203 AUUCdTdTUdTdT 241 D-2772 7476 GGUGUGGGGAAGCAU 35 7477 UUUAAUGCUUCCCCACAC 204UAACdTdT CdTdT 261 D-2773 7478 GACUGACUGAAGGCC 36 7479UGCAGGCCUUCAGUCAGU 205 UGCCdTdT CdTdT 263 D-2774 7480 CUGACUGAAGGCCUG 377481 UAUGCAGGCCUUCAGUCA 206 CAUCdTdT GdTdT 264 D-2775 7482UGACUGAAGGCCUGC 38 7483 UCAUGCAGGCCUUCAGUC 207 AUGCdTdT AdTdT 268 D-27767484 UGAAGGCCUGCAUGG 39 7485 UAAUCCAUGCAGGCCUUC 208 AUUCdTdT AdTdT 269D-2777 7486 GAAGGCCUGCAUGGA 40 7487 UGAAUCCAUGCAGGCCUU 209 UUCCdTdTCdTdT 276 D-2778 7488 UGCAUGGAUUCCAUG 41 7489 UGAACAUGGAAUCCAUGC 210UUCCdTdT AdTdT 278 D-2779 7490 CAUGGAUUCCAUGUU 42 7491UAUGAACAUGGAAUCCAU 211 CAUCdTdT GdTdT 281 D-2780 7492 GGAUUCCAUGUUCAU 437493 UCUCAUGAACAUGGAAUC 212 GAGCdTdT CdTdT 284 D-2781 7494UUCCAUGUUCAUGAG 44 7495 UAAACUCAUGAACAUGGA 213 UUUCdTdT AdTdT 290 D-27827496 GUUCAUGAGUUUGGA 45 7497 UAUCUCCAAACUCAUGAA 214 GAUCdTdT CdTdT 291D-2783 7498 UUCAUGAGUUUGGAG 46 7499 UUAUCUCCAAACUCAUGA 215 AUACdTdTAdTdT 295 D-2784 7500 UGAGUUUGGAGAUAA 47 7501 UGUAUUAUCUCCAAACUC 216UACCdTdT AdTdT 296 D-2785 7502 GAGUUUGGAGAUAAU 48 7503UUGUAUUAUCUCCAAACU 217 ACACdTdT CdTdT 316 D-2786 7504 AGGCUGUACCAGUGC 497505 UCCUGCACUGGUACAGCC 218 AGGCdTdT UdTdT 317 D-2787 7506GGCUGUACCAGUGCA 50 7507 UACCUGCACUGGUACAGC 219 GGUCdTdT CdTdT 329 D-27887508 GCAGGUCCUCACUUU 51 7509 UAUUAAAGUGAGGACCUG 220 AAUCdTdT CdTdT 330D-2789 7510 CAGGUCCUCACUUUA 52 7511 UGAUUAAAGUGAGGACCU 221 AUCCdTdTGdTdT 337 D-2790 7512 UCACUUUAAUCCUCU 53 7513 UGAUAGAGGAUUAAAGUG 222AUCCdTdT AdTdT 350 D-2791 7514 CUAUCCAGAAAACAC 54 7515UACCGUGUUUUCUGGAUA 223 GGUCdTdT GdTdT 351 D-2792 7516 UAUCCAGAAAACACG 557517 UCACCGUGUUUUCUGGAU 224 GUGCdTdT AdTdT 352 D-2793 7518AUCCAGAAAACACGG 56 7519 UCCACCGUGUUUUCUGGA 225 UGGCdTdT UdTdT 354 D-27947520 CCAGAAAACACGGUG 57 7521 UGCCCACCGUGUUUUCUG 226 GGCCdTdT GdTdT 357D-2795 7522 GAAAACACGGUGGGC 58 7523 UUUGGCCCACCGUGUUUU 227 CAACdTdTCdTdT 358 D-2796 7524 AAAACACGGUGGGCC 59 7525 UUUUGGCCCACCGUGUUU 228AAACdTdT UdTdT 364 D-2797 7526 CGGUGGGCCAAAGGA 60 7527UUCAUCCUUUGGCCCACC 229 UGACdTdT GdTdT 375 D-2798 7528 AGGAUGAAGAGAGGC 617529 UCAUGCCUCUCUUCAUCC 230 AUGCdTdT UdTdT 378 D-2799 7530AUGAAGAGAGGCAUG 62 7531 UCAACAUGCCUCUCUUCA 231 UUGCdTdT UdTdT 383 D-28007532 GAGAGGCAUGUUGGA 63 7533 UGUCUCCAACAUGCCUCU 232 GACCdTdT CdTdT 384D-2801 7534 AGAGGCAUGUUGGAG 64 7535 UAGUCUCCAACAUGCCUC 233 ACUCdTdTUdTdT 390 D-2802 7536 AUGUUGGAGACUUGG 65 7537 UUGCCCAAGUCUCCAACA 234GCACdTdT UdTdT 392 D-2803 7538 GUUGGAGACUUGGGC 66 7539UAUUGCCCAAGUCUCCAA 235 AAUCdTdT CdTdT 395 D-2804 7540 GGAGACUUGGGCAAU 677541 UCACAUUGCCCAAGUCUC 236 GUGCdTdT CdTdT 404 D-2805 7542GGCAAUGUGACUGCU 68 7543 UGUCAGCAGUCACAUUGC 237 GACCdTdT CdTdT 406 D-28067544 CAAUGUGACUGCUGA 69 7545 UUUGUCAGCAGUCACAUU 238 CAACdTdT GdTdT 417D-2807 7546 CUGACAAAGAUGGUG 70 7547 UCCACACCAUCUUUGUCA 239 UGGCdTdTGdTdT 418 D-2808 7548 UGACAAAGAUGGUGU 71 7549 UGCCACACCAUCUUUGUC 240GGCCdTdT AdTdT 469 D-2809 7550 CUCAGGAGACCAUUG 72 7551UAUGCAAUGGUCUCCUGA 241 CAUCdTdT GdTdT 470 D-2810 7552 UCAGGAGACCAUUGC 737553 UGAUGCAAUGGUCUCCUG 242 AUCCdTdT AdTdT 475 D-2811 7554AGACCAUUGCAUCAU 74 7555 UCCAAUGAUGCAAUGGUC 243 UGGCdTdT UdTdT 476 D-28127556 GACCAUUGCAUCAUU 75 7557 UGCCAAUGAUGCAAUGGU 244 GGCCdTdT CdTdT 480D-2813 7558 AUUGCAUCAUUGGCC 76 7559 UUGCGGCCAAUGAUGCAA 245 GCACdTdTUdTdT 487 D-2814 7560 CAUUGGCCGCACACU 77 7561 UACCAGUGUGCGGCCAAU 246GGUCdTdT GdTdT 494 D-2815 7562 CGCACACUGGUGGUC 78 7563UAUGGACCACCAGUGUGC 247 CAUCdTdT GdTdT 496 D-2816 7564 CACACUGGUGGUCCA 797565 UUCAUGGACCACCAGUGU 248 UGACdTdT GdTdT 497 D-2817 7566ACACUGGUGGUCCAU 80 7567 UUUCAUGGACCACCAGUG 249 GAACdTdT UdTdT 501 D-28187568 UGGUGGUCCAUGAAA 81 7569 UCUUUUUCAUGGACCACC 250 AAGCdTdT AdTdT 504D-2819 7570 UGGUCCAUGAAAAAG 82 7571 UCUGCUUUUUCAUGGACC 251 CAGCdTdTAdTdT 515 D-2820 7572 AAAGCAGAUGACUUG 83 7573 UGCCCAAGUCAUCUGCUU 252GGCCdTdT UdTdT 518 D-2821 7574 GCAGAUGACUUGGGC 84 7575UUUUGCCCAAGUCAUCUG 253 AAACdTdT CdTdT 522 D-2822 7576 AUGACUUGGGCAAAG 857577 UCACCUUUGCCCAAGUCA 254 GUGCdTdT UdTdT 523 D-2823 7578UGACUUGGGCAAAGG 86 7579 UCCACCUUUGCCCAAGUC 255 UGGCdTdT AdTdT 524 D-28247580 GACUUGGGCAAAGGU 87 7581 UUCCACCUUUGCCCAAGU 256 GGACdTdT CdTdT 552D-2825 7582 GUACAAAGACAGGAA 88 7583 UCGUUUCCUGUCUUUGUA 257 ACGCdTdTCdTdT 554 D-2826 7584 ACAAAGACAGGAAAC 89 7585 UAGCGUUUCCUGUCUUUG 258GCUCdTdT UdTdT 555 D-2827 7586 CAAAGACAGGAAACG 90 7587UCAGCGUUUCCUGUCUUU 259 CUGCdTdT GdTdT 562 D-2828 7588 AGGAAACGCUGGAAG 917589 UCGACUUCCAGCGUUUCC 260 UCGCdTdT UdTdT 576 D-2829 7590GUCGUUUGGCUUGUG 92 7591 UCACCACAAGCCAAACGA 261 GUGCdTdT CdTdT 577 D-28307592 UCGUUUGGCUUGUGG 93 7593 UACACCACAAGCCAAACG 262 UGUCdTdT AdTdT 578D-2831 7594 CGUUUGGCUUGUGGU 94 7595 UUACACCACAAGCCAAAC 263 GUACdTdTGdTdT 579 D-2832 7596 GUUUGGCUUGUGGUG 95 7597 UUUACACCACAAGCCAAA 264UAACdTdT CdTdT 581 D-2833 7598 UUGGCUUGUGGUGUA 96 7599UAAUUACACCACAAGCCA 265 AUUCdTdT AdTdT 583 D-2834 7600 GGCUUGUGGUGUAAU 977601 UCCAAUUACACCACAAGC 266 UGGCdTdT CdTdT 584 D-2835 7602GCUUGUGGUGUAAUU 98 7603 UCCCAAUUACACCACAAG 267 GGGCdTdT CdTdT 585 D-28367604 CUUGUGGUGUAAUUG 99 7605 UUCCCAAUUACACCACAA 268 GGACdTdT GdTdT 587D-2837 7606 UGUGGUGUAAUUGGG 100 7607 UGAUCCCAAUUACACCAC 269 AUCCdTdTAdTdT 588 D-2838 7608 GUGGUGUAAUUGGGA 101 7609 UCGAUCCCAAUUACACCA 270UCGCdTdT CdTdT 589 D-2839 7610 UGGUGUAAUUGGGAU 102 7611UGCGAUCCCAAUUACACC 271 CGCCdTdT AdTdT 593 D-2840 7612 GUAAUUGGGAUCGCC103 7613 UUUGGGCGAUCCCAAUUA 272 CAACdTdT CdTdT 594 D-2841 7614UAAUUGGGAUCGCCC 104 7615 UAUUGGGCGAUCCCAAUU 273 AAUCdTdT AdTdT 595D-2842 7616 AAUUGGGAUCGCCCA 105 7617 UUAUUGGGCGAUCCCAAU 274 AUACdTdTUdTdT 596 D-2843 7618 AUUGGGAUCGCCCAA 106 7619 UUUAUUGGGCGAUCCCAA 275UAACdTdT UdTdT 597 D-2844 7620 UUGGGAUCGCCCAAU 107 7621UUUUAUUGGGCGAUCCCA 276 AAACdTdT AdTdT 598 D-2845 7622 UGGGAUCGCCCAAUA108 7623 UGUUUAUUGGGCGAUCCC 277 AACCdTdT AdTdT 599 D-2846 7624GGGAUCGCCCAAUAA 109 7625 UUGUUUAUUGGGCGAUCC 278 ACACdTdT CdTdT 602D-2847 7626 AUCGCCCAAUAAACA 110 7627 UGAAUGUUUAUUGGGCGA 279 UUCCdTdTUdTdT 607 D-2848 7628 CCAAUAAACAUUCCC 111 7629 UCAAGGGAAUGUUUAUUG 280UUGCdTdT GdTdT 608 D-2849 7630 CAAUAAACAUUCCCU 112 7631UCCAAGGGAAUGUUUAUU 281 UGGCdTdT GdTdT 609 D-2850 7632 AAUAAACAUUCCCUU113 7633 UUCCAAGGGAAUGUUUAU 282 GGACdTdT UdTdT 610 D-2851 7634AUAAACAUUCCCUUG 114 7635 UAUCCAAGGGAAUGUUUA 283 GAUCdTdT UdTdT 611D-2852 7636 UAAACAUUCCCUUGG 115 7637 UCAUCCAAGGGAAUGUUU 284 AUGCdTdTAdTdT 612 D-2853 7638 AAACAUUCCCUUGGA 116 7639 UACAUCCAAGGGAAUGUU 285UGUCdTdT UdTdT 613 D-2854 7640 AACAUUCCCUUGGAU 117 7641UUACAUCCAAGGGAAUGU 286 GUACdTdT UdTdT 616 D-2855 7642 AUUCCCUUGGAUGUA118 7643 UGACUACAUCCAAGGGAA 287 GUCCdTdT UdTdT 621 D-2856 7644CUUGGAUGUAGUCUG 119 7645 UCCUCAGACUACAUCCAA 288 AGGCdTdT GdTdT 633D-2857 7646 CUGAGGCCCCUUAAC 120 7647 UUGAGUUAAGGGGCCUCA 289 UCACdTdTGdTdT 635 D-2858 7648 GAGGCCCCUUAACUC 121 7649 UGAUGAGUUAAGGGGCCU 290AUCCdTdT CdTdT 636 D-2859 7650 AGGCCCCUUAACUCA 122 7651UAGAUGAGUUAAGGGGCC 291 UCUCdTdT UdTdT 639 D-2860 7652 CCCCUUAACUCAUCU123 7653 UAACAGAUGAGUUAAGGG 292 GUUCdTdT GdTdT 640 D-2861 7654CCCUUAACUCAUCUG 124 7655 UUAACAGAUGAGUUAAGG 293 UUACdTdT GdTdT 641D-2862 7656 CCUUAACUCAUCUGU 125 7657 UAUAACAGAUGAGUUAAG 294 UAUCdTdTGdTdT 642 D-2863 7658 CUUAACUCAUCUGUU 126 7659 UGAUAACAGAUGAGUUAA 295AUCCdTdT GdTdT 643 D-2864 7660 UUAACUCAUCUGUUA 127 7661UGGAUAACAGAUGAGUUA 296 UCCCdTdT AdTdT 644 D-2865 7662 UAACUCAUCUGUUAU128 7663 UAGGAUAACAGAUGAGUU 297 CCUCdTdT AdTdT 645 D-2866 7664AACUCAUCUGUUAUC 129 7665 UCAGGAUAACAGAUGAGU 298 CUGCdTdT UdTdT 654D-2867 7666 GUUAUCCUGCUAGCU 130 7667 UUACAGCUAGCAGGAUAA 299 GUACdTdTCdTdT 660 D-2868 7668 CUGCUAGCUGUAGAA 131 7669 UCAUUUCUACAGCUAGCA 300AUGCdTdT GdTdT 661 D-2869 7670 UGCUAGCUGUAGAAA 132 7671UACAUUUCUACAGCUAGC 301 UGUCdTdT AdTdT 666 D-2870 7672 GCUGUAGAAAUGUAU133 7673 UAGGAUACAUUUCUACAG 302 CCUCdTdT CdTdT 667 D-2871 7674CUGUAGAAAUGUAUC 134 7675 UCAGGAUACAUUUCUACA 303 CUGCdTdT GdTdT 668D-2872 7676 UGUAGAAAUGUAUCC 135 7677 UUCAGGAUACAUUUCUAC 304 UGACdTdTAdTdT 669 D-2873 7678 GUAGAAAUGUAUCCU 136 7679 UAUCAGGAUACAUUUCUA 305GAUCdTdT CdTdT 673 D-2874 7680 AAAUGUAUCCUGAUA 137 7681UGUUUAUCAGGAUACAUU 306 AACCdTdT UdTdT 677 D-2875 7682 GUAUCCUGAUAAACA138 7683 UUAAUGUUUAUCAGGAUA 307 UUACdTdT CdTdT 692 D-2876 7684UUAAACACUGUAAUC 139 7685 UUAAGAUUACAGUGUUUA 308 UUACdTdT AdTdT 698D-2877 7686 ACUGUAAUCUUAAAA 140 7687 UCACUUUUAAGAUUACAG 309 GUGCdTdTUdTdT 699 D-2878 7688 CUGUAAUCUUAAAAG 141 7689 UACACUUUUAAGAUUACA 310UGUCdTdT GdTdT 700 D-2879 7690 UGUAAUCUUAAAAGU 142 7691UUACACUUUUAAGAUUAC 311 GUACdTdT AdTdT 701 D-2880 7692 GUAAUCUUAAAAGUG143 7693 UUUACACUUUUAAGAUUA 312 UAACdTdT CdTdT 706 D-2881 7694CUUAAAAGUGUAAUU 144 7695 UCACAAUUACACUUUUAA 313 GUGCdTdT GdTdT 749D-2882 7696 UACCUGUAGUGAGAA 145 7697 UAGUUUCUCACUACAGGU 314 ACUCdTdTAdTdT 770 D-2883 7698 UUAUGAUCACUUGGA 146 7699 UUCUUCCAAGUGAUCAUA 315AGACdTdT AdTdT 772 D-2884 7700 AUGAUCACUUGGAAG 147 7701UAAUCUUCCAAGUGAUCA 316 AUUCdTdT UdTdT 775 D-2885 7702 AUCACUUGGAAGAUU148 7703 UACAAAUCUUCCAAGUGA 317 UGUCdTdT UdTdT 781 D-2886 7704UGGAAGAUUUGUAUA 149 7705 UAACUAUACAAAUCUUCC 318 GUUCdTdT AdTdT 800D-2887 7706 UAUAAAACUCAGUUA 150 7707 UUUUUAACUGAGUUUUAU 319 AAACdTdTAdTdT 804 D-2888 7708 AAACUCAGUUAAAAU 151 7709 UGACAUUUUAACUGAGUU 320GUCCdTdT UdTdT 819 D-2889 7710 GUCUGUUUCAAUGAC 152 7711UCAGGUCAUUGAAACAGA 321 CUGCdTdT CdTdT 829 D-2890 7712 AUGACCUGUAUUUUG153 7713 UUGGCAAAAUACAGGUCA 322 CCACdTdT UdTdT 832 D-2891 7714ACCUGUAUUUUGCCA 154 7715 UGUCUGGCAAAAUACAGG 323 GACCdTdT UdTdT 833D-2892 7716 CCUGUAUUUUGCCAG 155 7717 UAGUCUGGCAAAAUACAG 324 ACUCdTdTGdTdT 851 D-2893 7718 UAAAUCACAGAUGGG 156 7719 UAUACCCAUCUGUGAUUU 325UAUCdTdT AdTdT 854 D-2894 7720 AUCACAGAUGGGUAU 157 7721UUUAAUACCCAUCUGUGA 326 UAACdTdT UdTdT 855 D-2895 7722 UCACAGAUGGGUAUU158 7723 UUUUAAUACCCAUCUGUG 327 AAACdTdT AdTdT 857 D-2896 7724ACAGAUGGGUAUUAA 159 7725 UAGUUUAAUACCCAUCUG 328 ACUCdTdT UdTdT 858D-2897 7726 CAGAUGGGUAUUAAA 160 7727 UAAGUUUAAUACCCAUCU 329 CUUCdTdTGdTdT 859 D-2898 7728 AGAUGGGUAUUAAAC 161 7729 UCAAGUUUAAUACCCAUC 330UUGCdTdT UdTdT 861 D-2899 7730 AUGGGUAUUAAACUU 162 7731UGACAAGUUUAAUACCCA 331 GUCCdTdT UdTdT 869 D-2900 7732 UAAACUUGUCAGAAU163 7733 UGAAAUUCUGACAAGUUU 332 UUCCdTdT AdTdT 891 D-2901 7734UCAUUCAAGCCUGUG 164 7735 UAUUCACAGGCUUGAAUG 333 AAUCdTdT AdTdT 892D-2902 7736 CAUUCAAGCCUGUGA 165 7737 UUAUUCACAGGCUUGAAU 334 AUACdTdTGdTdT 906 D-2903 7738 AAUAAAAACCCUGUA 166 7739 UCCAUACAGGGUUUUUAU 335UGGCdTdT UdTdT 907 D-2904 7740 AUAAAAACCCUGUAU 167 7741UGCCAUACAGGGUUUUUA 336 GGCCdTdT UdTdT 912 D-2905 7742 AACCCUGUAUGGCAC168 7743 UUAAGUGCCAUACAGGGU 337 UUACdTdT UdTdT 913 D-2906 7744ACCCUGUAUGGCACU 169 7745 UAUAAGUGCCAUACAGGG 338 UAUCdTdT UdTdT 934D-2907 7746 GAGGCUAUUAAAAGA 170 7747 UGAUUCUUUUAAUAGCCU 339 AUCCdTdTCdTdT 944 D-2908 7748 AAAGAAUCCAAAUUC 171 7749 UUUUGAAUUUGGAUUCUU 340AAACdTdT UdTdT 947 D-2909 7750 GAAUCCAAAUUCAAA 172 7751UUAGUUUGAAUUUGGAUU 341 CUACdTdT CdTdTSOD1 siRNA Synthesis

Oligoribonucleotides were assembled on an ABI 3900 synthesizer (AppliedBiosystems) according to the phosphoramidite oligomerization chemistry.The solid support was polystyrene loaded with 2′-deoxy-thymidine(purchased from Glen Research, Sterling, Va., USA) to give a synthesisscale of 0.2 μmol. Ancillary synthesis reagents, DNA and RNAphosphoramidites were obtained from SAFC Proligo (Hamburg, Germany).Specifically,5′-O-(4,4′-dimethoxytrityl)-3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite monomers of uridine (U), thymidine (dT),4-N-acetylcytidine (C^(Ac)), 6-N-benzoyladenosine (A^(bz)) and2-N-isobutyrlguanosine (G^(iBu)) with 2′-O-t-butyldimethylsilyl wereused to build the oligomers sequence. Coupling time for allphosphoramidites (70 mM in Acetonitrile) was 3 min employing5-Ethylthio-1H-tetrazole (ETT) as activator (0.5 M in Acetonitrile).Sequences were synthesized with removal of the final dimetoxytritylprotecting group on the synthesizer (“DMT off” synthesis). Uponcompletion of the solid phase synthesis oligoribonucleotides werecleaved from the solid support and de-protected using a 1:1 (v/v)mixture of aqueous methylamine (40%) and methylamine in ethanol (33%).After 90 minutes at 45° C. the solution was diluted with N,N-Dimethylformamide (DMF) and triethylamine trihydrofluoride (TEA.HF) was added.After incubation at 45° C. for 2 hours the oligoribonucleotides wereprecipitated with 1 M NaOAc and a mixture of acetone and ethanol 4:1(v/v). The pellets were dissolved in 1 M aqueous NaCl solution anddesalted by size exclusion chromatography. This was accomplished usingan AKTA Purifier HPLC System (GE Healthcare, Freiburg, Germany) equippedwith a HiTrap 5 mL column (GE Healthcare). Identity of theoligoribonucleotides was confirmed by MALDI mass spectrometry or ESImass spectrometry. To generate siRNAs from RNA single strands, equimolaramounts of complementary sense and antisense strands were mixed andannealed in a 20 mM NaCl, 4 mM sodium phosphate pH 6.8 buffer. siRNAswere stored frozen until use.

Example 2. In Vitro Screening of SOD1 siRNAs for Human SOD1 mRNASuppression

Human SOD1 targeting siRNAs (described in Table 3) were assayed forinhibition of endogenous SOD1 expression in HeLa cells, using the bDNA(branched DNA) assay to quantify SOD1 mRNA. Results from two dose assayswere used to select a subset of SOD1 dsRNA duplexes for dose responseexperiments in 4 types of cultured cells to calculate IC50's.

Cell Culture and Transfection

HeLa cells were obtained from ATCC (ATCC in Partnership with LGCStandards, Wesel, Germany) and cultured in HAM's F-12 Medium (BiochromGmbH, Berlin, Germany) supplemented to contain 10% fetal calf serum(Ultra-low IgG from GIBCO/Life Technologies) and 1% Pen/Strep (BiochromGmbH, Berlin, Germany) at 37° C. in an atmosphere with 5% CO₂ in ahumidified incubator.

For transfection with siRNA, HeLa cells were seeded at a density of19,000-20,000 cells/well in 96-well plates. Transfection of siRNA wascarried out with Lipofectamine 2000 (Invitrogen/Life Technologies)according to the manufacturer's instructions. For the two-dose screen,SOD1 siRNA concentrations of 1 nM or 0.1 nM were used. Dose responseexperiments were done with SOD1 siRNA concentrations of 10, 2.5, 0.6,0.16, 0.039, 0.0098, 0.0024, 0.0006, 0.00015, and 0.000038 nM. Controlwells were transfected with luciferase siRNA, Aha-1 siRNA, PLGF siRNA,or a control mix of unrelated siRNAs.

Branched DNA Assays—QuantiGene 2.0

After a 24-hour incubation with siRNA, media was removed and cells werelysed in 150 μl Lysis Mixture (1 volume lysis mixture, 2 volumesnuclease-free water) then incubated at 53° C. for 60 minutes. 80 μlWorking Probe Set SOD1 (gene target) and 90 μl Working Probe Set GAPDH(endogenous control) and 20 μl or 10 μl of cell-lysate were then addedto the Capture Plates. Capture Plates were incubated at 55° C. (forSOD1) and 53° C. (for GAPDH) (approx. 16-20 hrs). The next day, theCapture Plates were washed 3 times with at least 300 l of 1× Wash Buffer(nuclease-free water, Buffer Component 1 and Wash Buffer Component 2)(after the last wash, invert the plate and blot it against clean papertowels). 100 μl of pre-Amplifier Working Reagent was added to the SOD1Capture Plates, which were sealed with aluminum foil and incubated for 1hour at 55° C. Following a 1-hour incubation, the wash step wasrepeated, then 100 μl Amplifier Working Reagent was added to both SOD1and GAPDH capture plates. After 1 hour of incubation at 55° C. (SOD1) or53° C. (GAPDH), the wash and dry steps were repeated, and 100 μl LabelProbe was added. Capture plates were incubated at 50° C. (SOD1) or 53°C. (GAPDH) for 1 hour. The plates were then washed with 1× Wash Bufferand dried, and then 100 μl Substrate was added to the Capture Plates.Luminescence was read using 1420 Luminescence Counter (WALLAC VICTORLight, Perkin Elmer, Rodgau-Jügesheim, Germany) following 30 minutesincubation in the dark.

bDNA Data Analysis

For each SOD1 siRNA or control siRNA, four wells were transfected inparallel, and individual datapoints were collected from each well. Foreach well, the SOD1 mRNA level was normalized to the GAPDH mRNA level.The activity of a given SOD1 siRNA was expressed as percent SOD1 mRNAconcentration (normalized to GAPDH mRNA) in treated cells, relative tothe SOD1 mRNA concentration (normalized to GAPDH mRNA) averaged acrosscontrol wells.

Table 4 provides the results from the in vitro HeLa screen where theSOD1 siRNAs, the sequences of which are given in Table 3, were tested ateither 1 nM or 0.1 nM. The mean percentage of SOD1 mRNA (normalized toGAPDH mRNA) remaining in treated cells relative to controls, as well asthe standard deviation, is shown in Table 4 for each SOD1 siRNA. Anumber of SOD1 siRNAs at 1 nM were effective at reducing SOD1 mRNAlevels by more than 80% in HeLa cells. Furthermore, a number of SOD1siRNAs at 0.1 nM were effective at reducing SOD1 mRNA levels by morethan 80% in HeLa cells.

TABLE 4 Two dose results of in vitro screen of SOD1 siRNAs in HeLa cellsfor SOD1 gene expression inhibiting activity Remaining Remaining SOD1SOD1 mRNA [% of mRNA [% of Control] 24 Control] 24 hr After hr AftersiRNA 1 nM SOD1 SD 0.1 nM SOD1 SD duplex ID siRNA [%] siRNA [%] D-274187.2 2.7 70.6 3 D-2742 86.9 4.3 79.5 8.5 D-2743 89.6 3.6 80.6 8.8 D-274483.8 7.2 75.9 8.5 D-2745 95.1 9.1 84.1 6.8 D-2746 111.3 3.6 92.0 7.2D-2747 100.0 6.1 92.9 4.4 D-2748 100.4 3.1 91.6 12 D-2749 87.1 2.9 96.413 D-2750 94.2 7.1 93.1 8 D-2751 85.4 7.2 96.1 8 D-2752 27.2 3.6 70.26.5 D-2753 25.5 4.8 67.5 4.5 D-2754 23.2 4 70.2 2.3 D-2755 36.6 3.7 75.511 D-2756 9.1 0.7 29.2 2.6 D-2757 3.9 0.6 9.0 1.8 D-2758 6.4 1.1 13.92.8 D-2759 6.7 1.1 14.1 1 D-2760 32.3 3.4 61.9 8.8 D-2761 12.9 3.6 41.78.3 D-2762 16.9 2.6 41.2 10 D-2763 5.7 1.3 10.5 3.4 D-2764 9.2 2.7 19.54.9 D-2765 13.6 1.9 29.4 8.8 D-2766 8.7 1.1 28.1 6.6 D-2767 10.4 1.624.7 5.9 D-2768 13.0 1.4 27.7 7.3 D-2769 25.3 1.9 57.4 7.5 D-2770 14.91.6 35.5 4.4 D-2771 11.4 1.8 32.6 8.6 D-2772 10.6 1.3 27.9 4.7 D-277314.3 1.4 35.7 3.1 D-2774 7.1 1.3 23.0 1.5 D-2775 9.8 0.9 31.3 3.3 D-277611.1 2.9 31.3 5.3 D-2777 47.8 5.5 80.9 4.6 D-2778 7.4 0.6 26.5 4.2D-2779 7.9 0.6 17.9 3 D-2780 12.5 1.3 31.7 5.6 D-2781 16.3 2.3 39.1 8D-2782 10.2 3.1 25.4 3 D-2783 13.5 3.5 33.4 6.5 D-2784 12.3 2.5 36.3 5.4D-2785 14.6 3 30.5 7.4 D-2786 16.2 3.5 42.6 8 D-2787 14.4 4.2 37.3 6.5D-2788 9.8 3 21.6 6.6 D-2789 18.5 5.9 48.9 12 D-2790 11.6 3.8 28.1 5.6D-2791 8.9 1.8 26.6 5.6 D-2792 8.1 1.4 25.6 5.3 D-2793 9.3 1.6 26.6 3D-2794 8.9 1.9 25.8 4.2 D-2795 22.6 3.4 59.5 9.9 D-2796 15.1 0.7 43.01.9 D-2797 21.1 2.5 43.0 1.3 D-2798 10.4 1.2 28.0 5.1 D-2799 11.0 1.229.8 3.3 D-2800 21.3 2.4 52.4 4.7 D-2801 12.3 3.3 28.7 4 D-2802 8.4 1.818.8 3.7 D-2803 5.9 1 12.1 4.1 D-2804 11.8 1.6 28.9 7.5 D-2805 13.5 2.634.5 7.5 D-2806 5.5 1.1 10.4 2.5 D-2807 8.5 1.3 24.2 6.6 D-2808 9.5 1.526.0 1.4 D-2809 7.5 0.9 17.7 2.8 D-2810 12.1 2 43.1 8.3 D-2811 5.6 0.816.7 7 D-2812 14.2 1.4 42.5 8.2 D-2813 29.0 3.4 66.7 13 D-2814 35.7 3.573.4 15 D-2815 30.3 1.9 74.3 12 D-2816 14.6 2.1 47.2 5.1 D-2817 27.5 1.870.5 6.6 D-2818 9.6 0.8 32.9 7.2 D-2819 9.0 0.8 29.1 3 D-2820 10.8 1.438.7 3.5 D-2821 5.8 0.4 19.4 6.1 D-2822 10.5 2.5 46.3 6.8 D-2823 3.5 1.118.8 3.5 D-2824 9.9 3.2 43.8 0.8 D-2825 6.6 2.6 29.7 1.1 D-2826 8.0 1.940.6 7.2 D-2827 7.0 1.2 25.2 4.5 D-2828 6.4 2.2 22.4 1.7 D-2829 14.8 2.745.5 7.4 D-2830 9.4 2 28.5 6.5 D-2831 8.6 2.8 28.4 6.6 D-2832 12.3 3.243.4 3.2 D-2833 20.5 5.2 66.7 9.1 D-2834 10.7 2.5 35.9 2.2 D-2835 11.62.4 37.7 4 D-2836 24.1 3.3 57.0 4.2 D-2837 98.7 12 96.7 4.3 D-2838 20.54 49.5 1.4 D-2839 10.0 2.4 31.9 4.3 D-2840 50.2 8.3 89.2 7.4 D-2841 70.811 87.1 7.9 D-2842 79.7 21 90.9 3.6 D-2843 24.2 1.2 57.2 8.4 D-2844 21.56.4 51.4 1 D-2845 12.9 2.2 39.4 7.3 D-2846 10.2 2.6 30.5 2.6 D-2847 40.59.7 70.0 6.5 D-2848 41.8 7 63.7 6 D-2849 24.7 6.8 51.3 8.1 D-2850 79.47.5 76.5 16 D-2851 28.1 6.5 72.0 8.8 D-2852 13.8 2.1 56.9 4.8 D-285332.1 9.5 72.2 12 D-2854 21.5 3.9 58.8 10 D-2855 39.8 10 75.4 5.5 D-285614.4 3.4 40.4 5.8 D-2857 8.6 1 18.4 4.5 D-2858 10.1 1.1 19.1 4.8 D-285910.9 1.3 20.9 5.4 D-2860 7.4 1.3 11.7 3.8 D-2861 5.0 1.4 12.6 2.6 D-28625.5 1 13.8 2.7 D-2863 8.2 1.3 26.5 4.3 D-2864 9.1 1.6 40.2 3.4 D-28656.3 0.6 22.8 3.4 D-2866 7.0 1.7 17.8 4.3 D-2867 9.3 0.8 31.7 6.2 D-286810.3 2.5 30.8 6.5 D-2869 9.4 4.3 34.7 4.6 D-2870 5.9 0.6 18.1 2.6 D-28716.5 1.1 13.5 1.5 D-2872 10.5 1 31.3 5.3 D-2873 7.0 1.1 20.8 3.7 D-28749.4 2.4 35.3 5.7 D-2875 5.4 1.1 13.5 2.4 D-2876 14.1 4.6 45.9 5.2 D-287764.5 9.8 64.0 9 D-2878 57.0 14 62.9 8.1 D-2879 71.4 12 79.4 8.6 D-288079.7 11 100.9 4.9 D-2881 72.8 12 82.8 5.6 D-2882 64.4 8.8 73.2 6.9D-2883 80.1 4.9 86.3 13 D-2884 69.6 5.8 74.2 13 D-2885 76.9 2 76.7 18D-2886 74.0 0.7 80.4 3.4 D-2887 77.7 8.7 88.6 16 D-2888 70.3 5.1 66.22.2 D-2889 71.2 3 67.3 7.3 D-2890 75.3 7.9 71.2 6.4 D-2891 74.6 8.4 72.44.3 D-2892 72.5 6.9 71.6 5.7 D-2893 73.9 3.8 83.7 2.9 D-2894 66.9 5.772.4 4.9 D-2895 71.6 8.9 72.1 9 D-2896 71.0 5.6 74.4 1.3 D-2897 74.4 7.978.0 3.8 D-2898 74.0 5.8 73.5 1.6 D-2899 71.0 10 74.1 9.7 D-2900 71.34.1 77.8 5.8 D-2901 64.8 9.4 82.0 11 D-2902 53.6 5.2 82.7 15 D-2903 66.82.6 101.1 13 D-2904 62.6 7.8 87.5 20 D-2905 67.1 14 74.0 4.1 D-2906 64.03.2 73.9 12 D-2907 66.4 7.3 82.0 11 D-2908 72.6 20 85.2 23 D-2909 80.07.3 77.2 12

Twelve of the most active SOD1 siRNAs at 0.1 nM in HeLa cells wereevaluated in dose-response experiments. Table 5 provides the IC50concentrations resulting in 50% SOD1 mRNA suppression relative tocontrol for these twelve selected SOD1 siRNAs in HeLa cells. Thesetwelve SOD1 siRNAs were particularly potent in this experimentalparadigm, and exhibited IC50 values between 1 and 8 pM.

TABLE 5 IC50 results of in vitro assay of SOD1 siRNAs in HeLa cells forSOD1 gene expression inhibiting activity siRNA duplex ID IC50 Mean (pM)D-2757 1 D-2806 4 D-2860 2 D-2861 2 D-2875 4 D-2871 5 D-2758 5 D-2759 5D-2866 4 D-2870 4 D-2823 6 D-2858 8

The dose response data from HeLa cells used to identify the IC50s forthese twelve SOD1 siRNAs are presented in detail below in Table 6. Alltwelve siRNAs were determined to have pM IC50s in HeLa cells. The IC50data for the SOD1 siRNAs in Table 5 are a summary of the data presentedin Table 6 below.

TABLE 6 Dose response data for 12 SOD1 siRNAs in HeLa cells RemainingSOD1 mRNA (% of control) siRNA duplex 10 2.5 0.6 0.16 0.039 0.00980.0024 0.0006 0.00015 0.000038 IC50 ID nM nM nM nM nM nM nM nM nM nM(nM) D-2757 2 2 2 3 6 16 33 57 77 86 0.001 D-2806 2 3 3 6 13 32 59 83 90105 0.004 D-2860 5 5 5 6 10 22 50 68 87 92 0.002 D-2861 4 4 4 5 10 25 5173 81 92 0.002 D-2875 4 4 4 7 15 34 62 78 82 92 0.004 D-2871 4 5 4 8 1843 62 78 87 90 0.005 D-2758 5 5 5 9 17 41 70 81 97 111 0.005 D-2759 4 44 7 15 35 63 82 87 94 0.005 D-2866 3 3 4 8 17 39 54 79 80 76 0.004D-2870 4 5 5 8 18 41 59 77 93 101 0.004 D-2823 3 3 4 7 20 42 65 81 86 920.006 D-2858 5 5 5 9 21 46 72 82 88 94 0.008

Example 3. In Vitro Screen of Selected SOD1 siRNAs Against EndogenousSOD1 mRNA Expression in SH-SY5Y Cells, U87 Cells and Primary HumanAstrocytes

SH-SY5Y cells were obtained from ATCC (ATCC in Partnership with LGCStandards, Wesel, Germany) and cultured in Dulbecco's MEM (BiochromGmbH, Berlin, Germany) supplemented to contain 15% FCS (Ultra-low IgGfrom GIBCO/Life Technologies), 1% L-Glutamine (Biochrom GmbH, Berlin,Germany) and 1% Pen/Strep (Biochrom GmbH, Berlin, Germany) at 37° C. inan atmosphere with 5% CO₂ in a humidified incubator.

U87MG cells were obtained from ATCC (ATCC in Partnership with LGCStandards, Wesel, Germany) and cultured in ATCC-formulated Eagle'sMinimum Essential Medium (ATCC in Partnership with LGC Standards, Wesel,Germany) supplemented to contain 10% FCS (Ultra-low IgG from GIBCO/LifeTechnologies) and 1% Pen/Strep (Biochrom GmbH, Berlin, Germany) at 37°C. in an atmosphere with 5% CO₂ in a humidified incubator.

Primary human astrocytes were obtained from LONZA (Lonza Sales Ltd,Basel, Switzerland) and cultured in ABM Basal Medium (Lonza Sales Ltd,Basel, Switzerland) supplemented with AGM SingleQuot Kit (Lonza SalesLtd, Basel, Switzerland) at 37° C. in an atmosphere with 5% CO₂ in ahumidified incubator.

Transfection of SH-SY5Y cells, U87MG cells and primary human astrocyteswith twelve selected siRNAs (D-2757, D-2806, D-2860, D-2861, D-2875,D-2871, D-2758, D-2759, D-2866, D-2870, D-2823, D-2858), andquantitation of SOD1 and GAPDH mRNA levels with bDNA were performed in asimilar manner to that described for HeLa cells, except that thetransfection reagents were Lipofectamine2000 (Invitrogen/LifeTechnologies) for SH-SY5Y cells, RNAiMAX (Invitrogen/Life Technologies)for U87 cells, and Lipofectamine2000 (Invitrogen/Life Technologies) forprimary human astrocytes.

The dose response data from SH-SY5Y cells, U87MG cells and primary humanastrocytes used to identify the IC50s for these twelve SOD1 siRNAs(D-2757, D-2806, D-2860, D-2861, D-2875, D-2871, D-2758, D-2759, D-2866,D-2870, D-2823, D-2858), are presented in detail below in Tables 7, 8and 9, respectively. All twelve siRNAs were determined to have pM IC50sin U87 cells.

IC50 values are provided in Table 10. In primary human astrocytes, IC50swere higher than in SH-SY5Y and U87MG cells, in general.

TABLE 7 Dose response data for 12 SOD1 siRNAs in SH-SY5Y cells siRNARemaining SOD1 mRNA (% of control) duplex 10 2.5 0.6 0.16 0.039 0.00980.0024 0.0006 0.00015 0.000038 IC50 ID nM nM nM nM nM nM nM nM nM nM(nM) D-2757 8 13 16 22 36 55 72 92 107 114 0.013 D-2806 11 12 15 26 4071 103 121 117 131 0.025 D-2860 11 15 17 26 42 63 79 86 92 96 0.022D-2861 12 14 16 19 37 60 82 83 87 94 0.017 D-2875 20 25 35 59 79 92 9695 99 104 0.234 D-2871 15 19 23 42 71 87 95 94 99 96 0.103 D-2758 24 3536 58 91 96 134 123 105 94 0.369 D-2759 10 11 16 25 43 67 85 94 104 1080.026 D-2866 17 19 24 42 72 93 93 102 103 101 0.105 D-2870 19 22 26 4062 88 100 105 105 105 0.078 D-2823 11 16 25 47 64 84 91 98 105 95 0.099D-2858 16 21 25 46 68 91 92 95 103 116 0.106

TABLE 8 Dose response data for 12 SOD1 siRNAs in U87MG cells siRNARemaining SOD1 mRNA (% of control) duplex 10 2.5 0.6 0.16 0.039 0.00980.0024 0.0006 0.00015 0.000038 IC50 ID nM nM nM nM nM nM nM nM nM nM(nM) D-2757 3 4 3 4 5 8 19 50 86 99 0.001 D-2806 4 3 3 3 4 8 18 49 81106 0.001 D-2860 4 4 5 5 6 8 20 46 72 93 0.001 D-2861 5 6 6 6 8 15 39 6787 93 0.001 D-2875 4 5 5 5 6 9 19 45 76 99 0.001 D-2871 5 5 5 5 6 11 2450 77 86 0.001 D-2758 7 9 6 7 10 25 64 99 103 112 0.004 D-2759 6 6 5 6 821 50 80 93 104 0.002 D-2866 4 4 4 5 8 17 38 64 86 94 0.001 D-2870 5 5 55 7 7 13 31 63 85 0.003 D-2823 4 4 4 4 6 13 34 61 74 94 0.001 D-2858 7 66 7 8 14 33 54 71 94 0.001

TABLE 9 Dose response data for 12 SOD1 siRNAs in Primary HumanAstrocytes siRNA Remaining SOD1 mRNA (% of control) duplex 10 2.5 0.60.16 0.039 0.0098 0.0024 0.0006 0.00015 0.000038 IC50 ID nM nM nM nM nMnM nM nM nM nM (nM) D-2757 29 30 35 48 66 87 95 101 95 103 0.123 D-280626 32 35 47 63 78 87 95 95 98 0.113 D-2860 29 38 39 51 68 82 94 93 94101 0.192 D-2861 27 33 38 47 62 73 88 93 96 102 0.114 D-2875 25 28 39 4772 80 100 105 105 118 0.151 D-2871 25 34 42 52 63 83 97 100 97 108 0.182D-2758 27 29 31 41 51 71 86 91 95 98 0.049 D-2759 34 39 41 53 70 83 97101 98 103 0.219 D-2866 30 32 35 46 65 78 84 87 92 95 0.118 D-2870 34 3438 48 71 74 82 91 92 98 0.163 D-2823 27 31 40 53 67 80 84 86 92 97 0.186D-2858 29 30 37 55 72 91 93 100 104 104 0.197

The IC50 data for SOD1 siRNAs in Table 10 is a summary of the datapresented in Tables 7, 8 and 9.

TABLE 10 IC50 results of in vitro assays of SOD1 siRNAs in SH-SY5Ycells, U87MG cells and primary human astrocytes for SOD1 gene expressioninhibiting activity siRNA SH-SY5Y U87MG Primary Human duplex IC50 MeanIC50 Mean Astrocytes IC 50 ID (pM) (pM) Mean (pM) D-2757 13 1 123 D-280625 1 113 D-2860 22 1 192 D-2861 17 1 114 D-2875 234 1 151 D-2871 103 1182 D-2758 369 4 49 D-2759 26 2 219 D-2866 105 1 118 D-2870 78 3 163D-2823 99 1 186 D-2858 106 1 197

Example 4. siRNA Targeting SOD1

The passenger-guide strand duplexes of the SOD1 siRNA found to beefficacious are engineered into expression vectors and transfected intocells of the central nervous system or neuronal cell lines. Even thoughoverhang utilized in the siRNA knockdown study is a canonical dTdT forsiRNA, the overhang in the constructs may comprise any dinucleotideoverhang.

The cells used may be primary cells or derived from induced pluripotentstem cells (iPS cells).

SOD1 knockdown is then measured and deep sequencing performed todetermine the exact passenger and guide strand processed from eachconstruct administered in the expression vector.

A guide to passenger strand ratio is calculated to determine theefficiency of knockdown, e.g., of RNA Induced Silencing Complex (RISC)processing.

The N-terminus is sequenced to determine the cleavage site and todetermine the percent homogeneous cleavage of the target. It is expectedthat cleavage will be higher than 90 percent.

HeLa cells are co-transfected in a parallel study to analyze in vitroknockdown of SOD1. A luciferase construct is used as a control todetermine off-target effects.

Deep sequencing is again performed.

Example 5. Passenger and Guide Sequences Targeting SOD1

According to the present invention, SOD1 siRNAs were designed. These aregiven in Tables 11A and 11B. The passenger and guide strands aredescribed in the tables. In Tables 11A and 11B, the “miR” component ofthe name of the sequence does not necessarily correspond to the sequencenumbering of miRNA genes (e.g., VOYmiR-101 is the name of the sequenceand does not necessarily mean that miR-101 is part of the sequence).

TABLE 11A Passenger and Guide Sequences (5′-3′) Duplex Passenger ASGuide Name ID SS ID Passenger SEQ ID ID Guide SEQ ID VOYpre-001_D -D-2910 7752 CAAUGUG 342 7753 UUUGU 343 2806_Starting ACUGCUG CAGCAconstruct (18 native ACAACCC GUCAC nucleotides and AUUGUposition 19 is C; 3′ U terminal CC dinucleotide) VOYpre-002_D- D-29117754 CAAUGUG 344 7753 UUUGU 343 2806_p19MMU ACUGCUG CAGCA(position 19 U to form ACAAUCC GUCAC mismatch) AUUGU U VOYpre-003_D-D-2912 7755 CAAUGUG 345 7753 UUUGU 343 2806_p19GUpair ACUGCUG CAGCA(position 19 is G to ACAAGCC GUCAC form GU pair) AUUGU U VOYpre-004_D-D-2913 7756 CAAUGUG 346 7753 UUUGU 343 2806_p19AUpair ACUGCUG CAGCA(position 19 is A to ACAAACC GUCAC form AU pair) AUUGU U VOYpre-005_D-D-2914 7757 CAAUGUG 347 7753 UUUGU 343 2806_CMM (central ACAGCUG CAGCAmismatch) ACAAACC GUCAC AUUGU U VOYpre-006_D- D-2915 7758 CAAUGUG 3487753 UUUGU 343 2806_p19DEL ACUGCUG CAGCA (position 19 deleted) ACAACCGUCAC AUUGU U VOYpre-007_D- D-2916 7759 CAAUGUG 349 7753 UUUGU 3432806_p19ADD ACUGCUG CAGCA (nucleotide added at ACAAUCC GUCACposition 19; addition is C AUUGU U; keep C and U terminal CCdinucleotide) VOYpre-008_D- D-2917 7752 CAAUGUG 342 7753 UUUGU 3432806_Uloop ACUGCUG CAGCA ACAACCC GUCAC AUUGU U VOYpre-009_D- D-2918 7752CAAUGUG 342 7753 UUUGU 343 2806_AUloop ACUGCUG CAGCA ACAACCC GUCAC AUUGUU VOYpre-010_D - D-2919 7760 CAAUGUG 350 7753 UUUGU 343 2806_mir-22-loopACUGCUG CAGCA ACAACAC GUCAC AUUGU U VOYmiR-101_pre- D-2923 7752 CAAUGUG342 7753 UUUGU 343 001 hsa-mir-155; D- ACUGCUG CAGCA 2806 ACAACCC GUCACAUUGU U VOYmiR-102_pre- D-2924 7752 CAAUGUG 342 7753 UUUGU 343001 Engineered; D- ACUGCUG CAGCA 2806; let-7b stem ACAACCC GUCAC AUUGU UVOYmiR-103_pre- D-2925 7754 CAAUGUG 344 7753 UUUGU 343002 Engineered; D- ACUGCUG CAGCA 2806_p19M MU; let- ACAAUCC GUCAC7b stem AUUGU U VOYmiR-104_pre- D-2926 7755 CAAUGUG 345 7753 UUUGU 343003 Engineered; D- ACUGCUG CAGCA 2806_p19GUpair; let- ACAAGCC GUCAC7b stem AUUGU U VOYmiR-105_pre- D-2927 7756 CAAUGUG 346 7753 UUUGU 343004 Engineered; D- ACUGCUG CAGCA 2806_p19AUpair; let- ACAAACC GUCAC7b stem AUUGU U VOYmiR-106_pre- D-2928 7757 CAAUGUG 347 7753 UUUGU 343005 Engineered; D- ACAGCUG CAGCA stem 2806_CMM; let-7b ACAAACC GUCACAUUGU U VOYmiR-107_pre- D-2929 7758 CAAUGUG 348 7753 UUUGU 343006 Engineered; D- ACUGCUG CAGCA 2806_p19DEL; let-7b ACAACC GUCAC stemAUUGU U VOYmiR-108_pre- D-2930 7765 CAAUGUG 355 7753 UUUGU 343007 Engineered; D- ACUGCUG CAGCA 2806_p19ADD; let-7b ACAAUCC GUCAC stemC AUUGU U VOYmiR-109_pre- D-2931 7752 CAAUGUG 342 7753 UUUGU 343008 Engineered; D- ACUGCUG CAGCA 2806_Uloop; let-7b ACAACCC GUCAC stemAUUGU U VOYmiR-110_pre- D-2932 7752 CAAUGUG 342 7753 UUUGU 343009 Engineered; D- ACUGCUG CAGCA 2806_AUloop; let-7b ACAACCC GUCAC stemAUUGU U VOYmiR-111_pre- D-2933 7760 CAAUGUG 350 7753 UUUGU 343010 Engineered; D- ACUGCUG CAGCA 2806_mir-22-loop; let- ACAACAC GUCAC7b stem AUUGU U VOYmiR-112_pre- D-2934 7752 CAAUGUG 342 7753 UUUGU 343001 Engineered; PD; ACUGCUG CAGCA D-2806; let-7b basal- ACAACCC GUCACstem instability AUUGU U VOYmiR-113_pre- D-2935 7754 CAAUGUG 344 7753UUUGU 343 002 Engineered; D- ACUGCUG CAGCA 2806_p19M MU; let- ACAAUCCGUCAC 7b basal-stem AUUGU instability U VOYmiR-114_pre- D-2936 7757CAAUGUG 347 7753 UUUGU 343 005 Engineered; D- ACAGCUG CAGCA2806_CMM; let-7b ACAAACC GUCAC basal-stem instability AUUGU UVOYmiR-115_pre- D-2937 7760 CAAUGUG 350 7753 UUUGU 343010 Engineered; D- ACUGCUG CAGCA 2806_mir-22-loop; let- ACAACAC GUCAC7b basal-stem AUUGU instability U VOYmiR-116_pre- D-2938 7755 CAAUGUG345 7753 UUUGU 343 003 Engineered; D- ACUGCUG CAGCA 2806_p19GUpair; let-ACAAGCC GUCAC 7b basal-stem AUUGU instability U VOYmiR-117_pre- D-29397766 CGACGAA 356 7767 UCGCA 357 001 Engineered; D- GGCCGUG CACGG2757; let-7b stem UGCGCCC CCUUC GUCGU U VOYmiR-118_pre- D-2940 7768UGACUUG 358 7769 UCCAC 359 001 Engineered; D- GGCAAAG CUUUG2823; let-7b stem GUGGCCC CCCAA GUCAU U VOYmiR-119_pre- D-2941 7770AACUCAU 360 7771 UCAGG 361 001 Engineered; D- CUGUUAU AUAAC2866; let-7b stem CCUGCCC AGAUG AGUUU U VOYmiR-127 D-2942 7752 CAAUGUG342 7753 UUUGU 343 ACUGCUG CAGCA ACAACCC GUCAC AUUGU U VOYmiR-102.860D-2943 7772 CCCCUUA 362 7773 UAACA 363 ACUCAUC GAUGA UGUUCCC GUUAA GGGGUU VOYmiR102.861 D-2944 7774 CCCUUAA 364 7775 UUAAC 365 CUCAUCU AGAUGGUUACCC AGUUA AGGGU U VOYmiR-102.866 D-2945 7776 AACUCAU 366 7771 UCAGG361 CUGUUAU AUAAC CUUGCCC AGAUG AGUUU U VOYmiR-102.870 D-2946 7777GCUGUGG 367 7778 UAGGA 368 AAAUGUA UACAU UCUUCCC UUCUA CAGCU UVOYmiR-102.823 D-2947 7779 UGACUUG 369 7769 UCCAC 359 GGCAAAG CUUUGGUGAGCC CCCAA GUCAU U VOYmiR-104.860 D-2948 7780 CCCCUUA 370 7773 UAACA363 ACUCAUC GAUGA UGUUGCC GUUAA GGGGU U VOYmiR-104.861 D-2949 7781CCCUUAA 371 7775 UUAAC 365 CUCAUCU AGAUG GUUAGCC AGUUA AGGGU UVOYmiR-104.866 D-2950 7782 AACUCAU 372 7771 UCAGG 361 CUGUUAU AUAACCUUAGCC AGAUG AGUUU U VOYmiR-104.870 D-2951 7783 GCUGUGG 373 7778 UAGGA368 AAAUGUA UACAU UCUUGCC UUCUA CAGCU U VOYmiR-104.823 D-2952 7784UGACUUG 374 7769 UCCAC 359 GGCAAAG CUUUG GUAGGCC CCCAA GUCAU UVOYmiR-109.860 D-2953 7772 CCCCUUA 362 7773 UAACA 363 ACUCAUC GAUGAUGUUCCC GUUAA GGGGU U VOYmiR-104.861 D-2954 7774 CCCUUAA 364 7775 UUAAC365 CUCAUCU AGAUG GUUACCC AGUUA AGGGU U VOYmiR-104.866 D-2955 7776AACUCAU 366 7771 UCAGG 361 CUGUUAU AUAAC CUUGCCC AGAUG AGUUU UVOYmiR-109.870 D-2956 7777 GCUGUGG 367 7778 UAGGA 368 AAAUGUA UACAUUCUUCCC UUCUA CAGCU U VOYmiR-109.823 D-2957 7779 UGACUUG 369 7769 UCCAC359 GGCAAAG CUUUG GUGAGCC CCCAA GUCAU U VOYmiR-114.860 D-2958 7785CCCCUUA 375 7773 UAACA 363 ACACAUC GAUGA UGUUACC GUUAA GGGGU UVOYmiR-114.861 D-2959 7786 CCCUUAA 376 7775 UUAAC 365 CUGAUCU AGAUGGUUAACC AGUUA AGGGU U VOYmiR-114.866 D-2960 7787 AACUCAU 377 7771 UCAGG361 CUCUUAU AUAAC CUUGCCC AGAUG AGUUU U VOYmiR-114.870 D-2961 7788GCUGUGG 378 7778 UAGGA 368 AAUUGUA UACAU UCUUGCC UUCUA CAGCU UVOYmiR-114.823 D-2962 7789 UGACUUG 379 7769 UCCAC 359 GGGAAAG CUUUGGUGAGCC CCCAA GUCAU U VOYmiR-116.860 D-2963 7780 CCCCUUA 370 7773 UAACA363 ACUCAUC GAUGA UGUUGCC GUUAA GGGGU U VOYmiR-116.861 D-2964 7781CCCUUAA 371 7775 UUAAC 365 CUCAUCU AGAUG GUUAGCC AGUUA AGGGU UVOYmiR-116.866 D-2965 7790 AACUCAU 380 7771 UCAGG 361 CUGUUAU AUAACCUUGGCC AGAUG AGUUU U VOYmiR-116.870 D-2966 7783 GCUGUGG 373 7778 UAGGA368 AAAUGUA UACAU UCUUGCC UUCUA CAGCU U VOYmiR-116.823 D-2967 7784UGACUUG 374 7769 UCCAC 359 GGCAAAG CUUUG GUAGGCC CCCAA GUCAU UVoymiR-127.860 D-2968 7791 CCCCUUA 381 7773 UAACA 363 ACUCAUU GAUGAUGUUCCC GUUAA GGGGU U VoymiR-127.861 D-2969 7774 CCCUUAA 364 7775 UUAAC365 CUCAUCU AGAUG GUUACCC AGUUA AGGGU U VoymiR-127.866 D-2970 7776AACUCAU 366 7771 UCAGG 361 CUGUUAU AUAAC CUUGCCC AGAUG AGUUU UVoymiR-127.870 D-2971 7777 GCUGUGG 367 7778 UAGGA 368 AAAUGUA UACAUUCUUCCC UUCUA CAGCU U VoymiR-127.823 D-2972 7792 UGACUUG 382 7769 UCCAC359 GGCAAAG CUUUG GUAGCCC CCCAA GUCAU U VOYmiR-120 D-2973 7793 CAAUGUG383 7794 UUUGU 384 ACUGCUG CAGCA ACAAA GUCAC AUUGU C

TABLE 11B Passenger and Guide Sequences (5′-3′) Duplex Passenger ASGuide Name ID SS ID Passenger SEQ ID ID Guide SEQ ID VOYpre-011_D-D-2920 7761 UUUGUCA 351 7762 CAAUG 352 2806_passenger- GCAGUCA UGACUguide strand swap CAUUGUC GCUGA with terminal 3′ C CAAAU on passenger Cstrand VOYpre-012_D- D-2921 7761 UUUGUCA 351 7763 CAAUG 3532806_passenger- GCAGUCA UGACU guide strand swap CAUUGUC GCUGAwith terminal 3′ C CAAUU on passenger C strand VOYpre -013_D- D-29227764 UUUGUCA 354 7762 CAAUG 352 2806_passenger- GCAGUCA UGACUguide strand swap CAUUGAC GCUGA with terminal 3′ C CAAAU on passenger Cstrand

Example 6. SOD1 siRNA Constructs in AAV-miRNA Vectors

The passenger-guide strand duplexes of the SOD1 siRNA listed in Table 11are engineered into AAV-miRNA expression vectors. The construct from ITRto ITR, recited 5′ to 3′, comprises a mutant ITR, a promoter (either aCMV, a U6 or the CB6 promoter (which includes a CMVie enhancer, a CBApromoter and an SV40 intron), the passenger and guide strand (with aloop between the passenger and guide strand, a 5′ flanking region beforethe passenger strand and a 3′ flanking region after the guide strand)from Table 11, a rabbit globin polyA and wild type ITR. In vitro and invivo studies are performed to test the efficacy of the AAV-miRNAexpression vectors.

Example 7. Activity of Constructs in HeLa Cells

Seven of the SOD1 siRNA constructs described in Example 6 (VOYmiR-103,VOYmiR-105, VOYmiR-108, VOYmiR-114, VOYmiR-119, VOYmiR-120, andVOYmiR-127) and a control of double stranded mCherry were transfected inHeLa to test the activity of the constructs.

A. Passenger and Guide Strand Activity

The seven SOD1 siRNA constructs and a control of double stranded mCherrywere transfected into HeLa cells. After 48 hours the endogenous mRNAexpression was evaluated. All seven of the SOD1 siRNA constructs showedhigh activity of the guide strand with 75-80% knock-down and low to noactivity of the passenger strand. Guide strands of the SOD1 siRNAcandidate vectors showed high activity, yielding 75-80% knockdown ofSOD1, while passenger strands demonstrated little to no activity.

B. Activity of Constructs on SOD1

The seven SOD1 siRNA constructs and a control of double stranded mCherry(dsCherry) were transfected into HeLa cells at a MOI of 1e4 vg/cell, 1e3vg/cell, or 1e2 vg/cell. After 72 hours the endogenous mRNA expressionwas evaluated. All seven of the SOD1 siRNA constructs showed efficientknock-down at 1e3 vg/cell. Most of the SOD1 siRNA constructs showed highactivity (75-80% knock-down) as shown in FIG. 1 .

Example 8. Activity of Constructs in HEK Cells

Thirty of the SOD1 siRNA constructs described in Example 6(VOYmiR-102.860, VOYmiR-102.861, VOYmiR-102.866, VOYmiR-102.870,VOYmiR-102.823, VOYmiR-104.860, VOYmiR-104.861, VOYmiR-104.866,VOYmiR-104.870, VOYmiR-104.823, VOYmiR-109.860, VOYmiR-109.861,VOYmiR-109.866, VOYmiR-109.870, VOYmiR-109.823, VOYmiR-114.860,VOYmiR-114.861, VOYmiR-114.866, VOYmiR-114.870, VOYmiR-114.823,VOYmiR-116.860, VOYmiR-116.861, VOYmiR-116.866, VOYmiR-116.870,VOYmiR-116.823, VOYmiR-127.860, VOYmiR-127.861, VOYmiR-127.866,VOYmiR-127.870, VOYmiR-127.823) and a control of VOYmiR-114 and doublestranded mCherry were transfected in cells to test the activity of theconstructs.

A. Passenger and Guide Strand Activity in HEK293

The thirty constructs and two controls were transfected into HEK293Tcells. After 24 hours the endogenous mRNA expression was evaluated. Mostof the constructs showed high activity of the guide strand (FIG. 2 ) andlow to no activity of the passenger strand (FIG. 3 ).

B. Passenger and Guide Strand Activity in HeLa

The thirty constructs and two controls were transfected into HeLa cells.After 48 hours the endogenous mRNA expression was evaluated. Most of theconstructs showed high activity of the guide strand (FIG. 4 ) and low tono activity of the passenger strand (FIG. 5 ).

C. HeLa and HEK293 Correlation

The knock-down of the thirty constructs were similar between the HeLaand HEK293 cells. The thirty constructs showed knock-down for the guidestrand for the constructs (See FIGS. 2 and 4 ). Most of the guidestrands of the constructs showed 70-90% knock-down.

D. Capsid Selection

The top constructs from the HeLa and HEK293 are packaged in AAVs andwill undergo HeLa infection. To determine the best AAV to package theconstructs, mCherry packaged in either AAV2 or AAV-DJ8 was infected intoHeLa cells at a MOI of 10 vg/cell, 1e2 vg/cell, 1e3 vg/cell, 1e4 vg/cellor 1e5 vg/cell and the expression was evaluated at 40 hours. AAV2 wasselected as the capsid to package the top constructs.

E. AAV2 Production

The top constructs from the HeLa and HEK293 are packaged in AAV2 (1.6kb) and a control of double stranded mCherry (dsmCherry) was alsopackaged. The packaged constructs underwent Idoixanol purification priorto analysis. The AAV titer is shown in Table 12.

TABLE 12 AAV Titer Construct AAV Titer (genomes per ul) VOYmir-102.8605.5E+08 VOYmir-102.861 1.0E+09 VOYmir-102.823 9.1E+08 VOYmir-104.8611.2E+09 VOYmir-104.866 8.0E+08 VOYmir-104.823 5.7E+08 VOYmir-109.8603.1E+08 VOYmir-109.861 8.9E+08 VOYmir-109.866 6.0E+08 VOYmir-109.8236.0E+08 VOYmir-114.860 4.7E+08 VOYmir-114.861 3.7E+08 VOYmir-114.8661.0E+09 VOYmir-144.823 1.7E+09 VOYmir-116.860 1.0E+09 VOYmir-116.8669.1E+08 VOYmir-127.860 1.2E+09 VOYmir-127.866 9.0E+08 dsmCherry 1.2E+09

The effect of transduction on SOD1 knock-down in HeLa cells is shown inFIG. 6 . In addition, in HeLa cells, a larger MOI (1.0E+04 compared to1.0E+05) did not show increased knock-down for every construct.

F. Activity of Constructs in Human Motor Neuron Progenitors (HMNPs)

The top 18 pri-miRNA constructs as described in Example 8E and a controlof mCherry were infected into human motor neuron progenitor (HMNP) cellsat a MOI of 10E5. After 48 hours the endogenous mRNA expression wasevaluated. About half of the constructs gave greater than 50% silencingof SOD1 in HMNPs and 4 of those gave greater than 70% silencing (FIG. 7).

G. Construct Selection for In Vivo Studies

The top twelve constructs are selected which had a major effect on thetarget sequence and a minor effect on the cassette. These constructspackaged in AAV-rh10 capsids are formulated for injection andadministered in mammals to study the in vivo effects of the constructs.

Example 9. In Vitro Study of Constructs

The 18 constructs and mCherry control described in Example 8D packagedin AAV2 were used for this study. For this study, HEK293T cells (FisherScientific, Cat. #HCL4517) in culture medium (500 ml of DMEM/F-12GLUTAMAX™ supplement (Life Technologies, Cat #. 10565-018), 50 ml FBS(Life Technologies, Cat #. 16000-044, lot:1347556), 5 ml MEMNon-essential amino acids solution (100×) (Cat. #11140-050) and 5 mlHEPES (1M) (Life Technologies, Cat #. 15630-080)), U251MG cells (P18)(Sigma, Cat #. 09063001-1VL) in culture medium (500 ml of DMEM/F-12GLUTAMAX™ supplement (Life Technologies, Cat #. 10565-018), 50 ml FBS(Life Technologies, Cat #. 16000-044, lot:1347556), 5 ml MEMNon-essential amino acids solution (100×) (Cat. #11140-050) and 5 mlHEPES (1M) (Life Technologies, Cat #. 15630-080)) or normal humanastrocyte (HA) (Lonza, Cat #CC-2565) in culture medium (ABM Basal Medium500 ml (Lonza, Cat #. CC-3186) supplemented with AGM SingleQuot KitSuppl. & Growth Factors (Lonza, Cat #. CC-4123)) were used to test theconstructs. HEK293T cells (5×10E4 cells/well in 96 well plate), U251MGcells (2×10E4 cells/well in 96 well plate) and HA cells (2×10E4cells/well in 96 well plate) were seeded and the MOI used for infectionof cells was 1.0E+05. After 48 hours the cells were analyzed and theresults are shown in Table 13.

TABLE 13 Relative SOD1 mRNA level Relative SOD1 mRNA Level (%)(Normalized to GAPDH) Construct HEK293T U251MG HA VOYmiR-102.823 19.549.6 87.3 VOYmiR-102.860 1.7 5.3 19.2 VOYmiR-102.861 1.1 13.9 42.6VOYmiR-104.823 49.9 69.6 102.7 VOYmiR-104.861 1.0 10.7 36.3VOYmiR-104.866 12.3 54.6 85.5 VOYmiR-109.823 23.0 46.1 84.6VOYmiR-109.860 1.9 8.3 35.6 VOYmiR-109.861 1.9 22.7 57.3 VOYmiR-109.8664.1 38.5 67.9 VOYmiR-114.823 19.3 44.7 82.3 VOYmiR-114.860 1.4 4.7 17.6VOYmiR-114.861 1.1 9.7 48.1 VOYmiR-114.866 4.0 38.7 78.2 VOYmiR-116.8601.1 4.8 15.8 VOYmiR-116.866 5.5 40.2 73.7 VOYmiR-127.860 1.0 2.1 7.4VOYmiR-127.866 1.0 15.4 43.8 mCherry 100.0 100.2 100.1

Greater than 80% knock-down was seen in the HEK293T cells for mostconstructs. More than half of the constructs showed greater than 80%knock-down in the U251MG cells and in the HA cells.

Example 10. Dose Dependent SOD1 Lowering

Four of the top 18 pri-miRNA constructs as described in Example 8E and acontrol of mCherry were transfected into a human astrocyte cell line(U251MG) or a primary human astrocyte (HA) at an MOI of 1.0E+02,1.0E+03, 1.0E+04, 1.0E+05 or 1.0E+06. After 48 hours the endogenous mRNAexpression was evaluated and the dose-dependent silencing are shown inFIG. 8 (U251MG) and FIG. 9 (HA). For all constructs, the increase indose also correlated to an increase in the amount of SOD1 mRNA that wasknocked-down.

Example 11. Time Course of SOD1 Knock-Down

Two pri-miRNA constructs (VOYmiR-120 and VOYmiR-122), a negative controland a positive control of SOD1 siRNA were transfected into a humanastrocyte cell line (U251MG). The relative SOD1 mRNA was determined for60 hours as shown in FIG. 10 . 70-75% knock-down of hSOD1 was seen forboth pri-miR constructs after Nucleofector transfection, with thegreatest knock-down seen in the 12-24 hour window.

Example 12. SOD1 Knock-Down and Stand Percentages

VOYmiR-104 was transfected into HeLa cells at a concentration of 50 pM,100 pM and 150 pM and compared to untreated (UT) cells. The relativeSOD1 mRNA, the percent of the guide strand and the percent of thepassenger strand was determined at 36, 72, 108 and 144 hours as shown inFIGS. 11A-11C. The highest concentration (150 pM) showed the greatestreduction in expression, but all three doses showed a significantreduction in the expression of SOD1.

Example 13. Constructs Targeting SOD1

Constructs were designed for Dog SOD1 and the constructs are given inTable 14. Dog SOD1 is 100% conserved with human in the region targetedin the present invention. The passenger and guide sequences aredescribed in the table. In Table 14, the “miR” component of the name ofthe sequence does not necessarily correspond to the sequence numberingof miRNA genes (e.g., dVOYmiR-102 is the name of the sequence and doesnot necessarily mean that miR-102 is part of the sequence).

TABLE 14 Dog sequences (5′-3′) Duplex SS Passenger AS Guide Name ID IDPassenger SEQ ID ID Guide SEQ ID dVOYmiR- D-2974 7795 GCAGGUCC 385 7796GAUUAAAG 386 102.788 UCACUUUA UGAGGACC AUGCC UGCUU dVOYmiR- D-2975 7797GGCAAUGU 387 7798 UGUCAGCA 388 102.805 GACUGCUG GUCACAUU ACCCC GCCUUdVOYmiR- D-2976 7799 GCAGGUCC 389 7796 GAUUAAAG 386 104.788 UCACUUUAUGAGGACC AUUCC UGCUU dVOYmiR- D-2977 7800 GGCAAUGU 390 7798 UGUCAGCA 388104.805 GACUGCUG GUCACAUU AUGCC GCCUU dVOYmiR- D-2978 7801 GCAGGUCC 3917796 GAUUAAAG 386 109.788 UCACUUUA UGAGGACC AUCCC UGCUU dVOYmiR- D-29797802 GGCAAUGU 392 7798 UGUCAGCA 388 109.805 GACUGCUG GUCACAUU AUACCGCCUU dVOYmiR- D-2980 7803 GCAGGUCC 393 7796 GAUUAAAG 386 114.788UGACUUUA UGAGGACC AUCCC UGCUU dVOYmiR- D-2981 7804 GGCAAUGU 394 7798UGUCAGCA 388 114.805 GUCUGCUG GUCACAUU AUACC GCCUU dVOYmiR- D-2982 7801GCAGGUCC 391 7796 GAUUAAAG 386 116.788 UCACUUUA UGAGGACC AUCCC UGCUUdVOYmiR- D-2983 7802 GGCAAUGU 392 7798 UGUCAGCA 388 116.805 GACUGCUGGUCACAUU AUACC GCCUU dVoymiR- D-2984 7801 GCAGGUCC 391 7805 GAUUAAAG 395127.788 UCACUUUA UGAGGACC AUCCC UGCUUU dVoymiR- D-2985 7802 GGCAAUGU 3927806 UGUCAGCA 396 127.805 GACUGCUG GUCACAUU AUACC GCCUUU

Example 14. Effect of the Position of Modulatory Polynucleotides

A. Effect on Viral Titers

A siRNA molecule (VOYmiR-114 or VOYmiR-126) was inserted into anexpression vector (genome size 2400 nucleotides; scAAV) at six differentlocations as shown in FIG. 12 . In FIG. 12 , “ITR” is the invertedterminal repeat, “I” represents intron, “P” is the polyA and “MP” is themodulatory polynucleotide comprising the siRNA molecule. The viraltiters were evaluated using TaqMan PCR for the 6 position and for acontrol (construct without a modulatory polynucleotide; scAAV) and theresults are shown in Table 15.

TABLE 15 Viral Titers siRNA siRNA Molecule Virus Titer Molecule Position(VG per 15-cm dish) VOYmiR-114 Position 1 5.5E+10 VOYmiR-114 Position 25.5E+10 VOYmiR-114 Position 3 4.5E+10 VOYmiR-114 Position 4 3.7E+10VOYmiR-114 Position 5 6.5E+10 VOYmiR-114 Position 6 2.5E+10 VOYmiR-126Position 1 1.6E+10 VOYmiR-126 Position 2 3.2E+10 VOYmiR-126 Position 36.0E+10 VOYmiR-126 Position 4 1.6E+10 VOYmiR-126 Position 5 9.5E+09VOYmiR-126 Position 6 6.0E+10 — Control 2.1E+11B. Effect on Genome Integrity

A siRNA molecule (VOYmiR-114) was inserted into an expression vector(genome size 2400 nucleotides; scAAV) at six different locations and acontrol without a modulatory polynucleotide (scAAV) as shown in FIG. 12. In FIG. 12 , “ITR” is the inverted terminal repeat, “I” representsintron, “P” is the polyA and “MP” is the modulatory polynucleotidecomprising the siRNA molecule. Viral genomes were extracted frompurified AAV preparations and run on a neutral agarose gel. Truncatedgenomes were seen in all constructs and the approximate percent of thetruncated genomes (percent of the total) is shown in Table 16.

TABLE 16 Truncated Genomes Construct % of total Position 1 50 Position 241 Position 3 49 Position 4 34 Position 5 33 Position 6 59 Control 9

Position 6 had the greatest number of truncated genomes with Position 4and 5 having the least amount of truncated genomes.

C. Effect on Knock-Down Efficiency

A siRNA molecule (VOYmiR-114) was inserted into an expression vector(AAV2) (genome size 2400 nucleotides; scAAV) at six different locationsas shown in FIG. 12 . In FIG. 12 , “ITR” is the inverted terminalrepeat, “I” represents intron, “P” is the polyA and “MP” is themodulatory polynucleotide comprising the siRNA molecule. Transduction ofHeLa cells was conducted at 1×10⁴ vg/cell, 1×10³ vg/cell and 1×10²vg/cell. The SOD1 mRNA expression (as % of control (eGFP)) wasdetermined 72 hours post-infection and the results are shown in Table17.

TABLE 17 SOD1 Expression SOD1 mRNA expression (% of control) Construct 1× 10⁴ vg/cell 1 × 10³ vg/cell 1 × 10² vg/cell Position 1 40 59 69Position 2 31 46 75 Position 3 50 66 81 Position 4 21 34 55 Position 549 52 67 Position 6 31 37 62 Control (eGFP) 100 100 94

Position 3 had the highest SOD1 mRNA expression (as % of control) andPosition 4 had the lowest SOD1 mRNA expression (as % of control).

Example 15. Effect of Genome Size

A. Effect on Viral Titers

A siRNA molecule (VOYmiR-114) was inserted into an expression vector(genome size 2 kb; scAAV) at positions 1, 2, 5 and 6 as shown in FIG. 12. In FIG. 12 , “ITR” is the inverted terminal repeat, “I” representsintron, “P” is the polyA and “MP” is the modulatory polynucleotidecomprising the siRNA molecule. A double stranded control without a siRNAmolecule (genome size 1.6 kb; scAAV ctrl) and a double strandedexpression vector (scAAV miR114; ITR (105 nucleotide)—Promoter (˜900nucleotides)—modulatory polynucleotide comprising the siRNA molecule(158 nucleotides)—polyA sequence (127 nucleotides) and ITR) was comparedas well as a control (eGFP; scAAV) with no siRNA molecule. The viraltiters were evaluated using TaqMan PCR and the results are shown inTable 18.

TABLE 18 Viral Titers Virus Titer (VG Construct Size per 15-cm dish)Position 1   2 kb 9.5E+10 Position 2   2 kb 1.2E+11 scAAV miR114 1.6 kb1.1E+11 Position 5   2 kb 2.4E+10 Position 6   2 kb 1.1E+11 Control   2kb 2.2E+11

The lowest viral titers were seen with the position 5 construct and thegreatest was with the position 2 construct.

B. Effect on Genome Integrity

A siRNA molecule (VOYmiR-114) was inserted into an expression vector(genome size 2 kb; scAAV) at positions 1, 2, 5 and 6 as shown in FIG. 12. In FIG. 12 , “ITR” is the inverted terminal repeat, “I” representsintron, “P” is the polyA and “MP” is the modulatory polynucleotidecomprising the siRNA molecule. A double stranded control without a siRNAmolecule (genome size 1.6 kb; scAAV ctrl) and a double strandedexpression vector (scAAV miR114; ITR (105 nucleotide)—Promoter (˜900nucleotides)—modulatory polynucleotide comprising the siRNA molecule(158 nucleotides)—polyA sequence (127 nucleotides) and ITR) was comparedas well as a control (eGFP; scAAV) with no siRNA molecule. Truncatedgenomes were seen in all constructs and the approximate percent of thetruncated genomes (percent of the total) is shown in Table 19.

TABLE 19 Truncated Genomes Construct Size % of total Position 1   2 kb34 Position 2   2 kb 30 scAAV miR114 1.6 kb 20 Position 5   2 kb 21Position 6   2 kb 46 Control   2 kb 5

All constructs were determined to have some truncated genomes.

An additional study was conducted to determine the effect of differentsiRNA molecules. VOYmiR-114 and VOYmiR-126 were inserted into separateexpression vectors (genome size 1.6 kb; scAAV) at position 3 as shown inFIG. 12 . In FIG. 12 , “ITR” is the inverted terminal repeat, “I”represents intron, “P” is the polyA and “MP” is the modulatorypolynucleotide comprising the siRNA molecule. For the VOYmiR-114construct the distance between the 5′ end of the vector genome (1526nucleotides) and the center of the modulatory polynucleotide (middle ofthe flexible loop) is 1115 nucleotides. For the VOYmiR-126 construct thedistance between the 5′ end of the vector genome (1626 nucleotides) andthe center of the modulatory polynucleotide (middle of the flexibleloop) is 1164 nucleotides.

For the VOYmiR-114 construct, the viral titer (VG per 15-cm dish) wasabout 1.1E+11. For the VOYmiR-126 construct, the intron probe viraltiter (VG per 15-cm dish) was about 1.2E+12. The control was about2.1E+11 (VG per 15-cm dish). VOYmir-114 had about 20% truncated genomes,VOYmiR-126 has about 15% truncated genomes and the control had about 5%truncated genomes.

Example 16. Effect of Single Stranded Constructs

A. Effect on Viral Titers

A siRNA polynucleotide (VOYmiR-114) was inserted into an expressionvector (genome size 4.7 kb; ssAAV) at positions 1, 3 and 5 as shown inFIG. 12 and there was a control also tested without a siRNApolynucleotide (genome size 2 kb; ssAAV). In FIG. 12 , “ITR” is theinverted terminal repeat, “I” represents intron, “P” is the polyA and“MP” is the modulatory polynucleotide comprising the siRNA molecule. Theviral titers were evaluated using TaqMan PCR and the results are shownin Table 20.

TABLE 20 Viral Titers Construct Virus Titer (VG per 15-cm dish) Position1 5.0E+11 Position 3 7.5E+11 Position 5 3.5E+11 Control 2.5E+11

Position 3 showed the greatest viral titers followed by position 1 andthen position 5.

B. Effect on Genome Integrity

A siRNA molecule (VOYmiR-114) was inserted into an expression vector(genome size 4.7 kb; ssAAV) at positions 1, 3 and 5 as shown in FIG. 12and there was a control also tested without a modulatory polynucleotide(genome size 2 kb; ssAAV). In FIG. 12 , “ITR” is the inverted terminalrepeat, “I” represents intron, “P” is the polyA and “MP” is themodulatory polynucleotide comprising the siRNA molecule. Viral genomeswere extracted from purified AAV preparations and run on a neutralagarose gel. Truncated genomes were seen in all constructs and theapproximate percent of the truncated genomes (percent of the total) isshown in Table 21.

TABLE 21 Truncated Genomes Construct % of total Position 1 48 Position 330 Position 5 72 Control 0

Position 5 had the greatest number of truncated genomes with Position 3having the least amount of truncated genomes.

C. Effect on Knock-Down Efficiency

A siRNA molecule (VOYmiR-114) was inserted into an expression vector(genome size 4.7 kb; ssAAV) at positions 1, 3 and 5 as shown in FIG. 12and there was a single stranded control without a siRNA molecule (genomesize 2 kb; ssAAV ctrl), a double stranded control without a siRNAmolecule (genome size 1.6 kb; scAAV ctrl) and a double strandedexpression vector (genome size 2.4 kb; scAAV miR114) with a siRNAmolecule. In FIG. 12 , “ITR” is the inverted terminal repeat, “I”represents intron, “P” is the polyA and “MP” is the modulatorypolynucleotide comprising the siRNA molecule. Transduction of HeLa cellswas conducted at 1×10⁴ vg/cell, 1×10³ vg/cell and 1×10² vg/cell. TheSOD1 mRNA expression (as % of control (eGFP)) was determined 72 hourspost-infection and the results are shown in Table 22.

TABLE 22 SOD1 Expression SOD1 mRNA expression (% of control) Construct 1× 10⁴ vg/cell 1 × 10³ vg/cell 1 × 10² vg/cell Position 1 62 85 87Position 3 77 93 99 Position 5 59 82 84 ssAAV ctrl 100 101 108 scAAVctrl 95 97 102 scAAV miR114 23 33 62

Position 3 had the highest SOD1 mRNA expression (as % of control), thenposition 1 and the single stranded constructs with the lowest SOD1 mRNAexpression (as % of control) was Position 5. None of the single strandedconstructs had knock-down efficiency that was as low as the doublestranded control with a siRNA polynucleotide.

Example 17. SOD1 Knock-Down In Vivo

To evaluate the in vivo biological activity of pri-miRNAs,self-complementary pri-miRNAs (VOYmiR-114.806, VOYmiR127.806,VOYmiR102.860, VOYmiR109.860, VOYmiR114.860, VOYmiR116.860,VOYmiR127.860, VOYmiR102.861, VOYmiR104.861, VOYmiR109.861,VOYmiR114.861, VOYmiR109.866, VOYmiR116.866, or VOYmiR127.866) arepackaged in AAV-DJ with a CBA promoter.

In mice, these packaged pri-miRNAs or a control of vehicle only(phosphate-buffered saline with 5% sorbitol and 0.001% F-68) wereadministered by a 10-minute intrastriatal infusion. Female or maleTg(SOD1)3Cje/J mice (Jackson Laboratory, Bar Harbor, Me.), which expresshuman SOD1, and of approximately 20-30 g body weight, receive unilateralinjections of 5 uL test article which is targeted to the striatum(anteroposterior+0.5 mm, mediolateral+2 mm, relative to bregma;dorsoventral 3.8 mm, relative to skull surface). Test articles areinjected (5 animals per test article) at 0.5 uL/min. using pre-filled,pump-regulated Hamilton micro-syringes (1701 model, 10 μl) with 33-gaugeneedles. At 1, 2, 3, 4 or 6 weeks following the injection, animals aresacrificed, brains are removed, and ipsilateral striata encompassing theinfusion site from a 1 mm coronal slab, as well as striatal tissue fromthe adjacent 1 mm coronal slabs are dissected and flash frozen. Mousetissue samples are lysed, and human SOD1 protein levels, and SOD1 andmouse GAPDH (mGAPDH) mRNA levels are quantified. SOD1 protein levels arequantified by ELISA (eBioscience (Affymetrix, San Diego, Calif.)), andtotal protein levels are quantified by BCA analysis (ThermoFisherScientific, Waltham, Mass.). For each tissue sample, the level of SOD1protein normalized to total protein is calculated as an average of 2determinations. These normalized SOD1 protein levels are furthernormalized to the vehicle group, then averaged to obtain a group(treatment) average. SOD1 and mGAPDH mRNA levels are quantified byqRT-PCR. For each tissue sample, the ratio of SOD1/mGAPDH (normalizedSOD1 mRNA level) is calculated as an average of 3 determinations. Theseratios are then averaged to obtain a group (treatment) average. Thesegroup averages are further normalized to the vehicle group.

In non-human primates, test articles (1×10¹³-3×10¹³ vg of pri-miRNApackaged in AAV-DJ with a CBA promoter) or vehicle are administered byintrathecal lumbar bolus. Female cynomolgus monkeys (Macacafascicularis, CR Research Model Houston, Houston, Tex.) of approximately2.5-8.5 kg body weight, receive implanted single intrathecal catheterswith the tip of the catheter located at the lumbar spine. Test articlesare administered (4 animals per test article) comprising three 1 mLbolus injections (1 mL/minute), at approximately 60-minute intervals. At4 to 6 weeks following the administration, animals are sacrificed, andselected tissues harvested for bioanalytical and histologicalevaluation. SOD1 protein and mRNA levels are assessed for suppressionafter treatment with pri-miRNA packaged in AAV-DJ with a CBA promoter,relative to the vehicle group.

Example 18. SOD1 Knock-Down In Vivo Using VOYmiR-114.806

In Tg(SOD1)3Cje/J mice, VOYmiR-114.806 packaged in AAVDJ with a CBApromoter as described in Example 17. The mice were administered byunilateral intrastriatal administration a dose of 3.7×10⁹ vg. After 1 or2 weeks, there was no significant reduction in normalized SOD1 proteinlevels; normalized SOD1 protein levels were 98±11% (standard deviation)and 98±10% of the vehicle control group after 1 and 2 weeks,respectively. By week 3, VOYmiR-114.806 reduced the normalized SOD1protein level to 84±9.0% of the vehicle control group, which wasstatistically significant (p<0.05, One-way ANOVA with Dunnett's post-hocanalysis). By weeks 4 and 6, VOYmiR-114.806 reduced the normalized SOD1protein level to 73±7.9% (p<0.0001) and 75±7.4% (p<0.0001),respectively, of the vehicle control group. These results demonstratethat VOYmiR-114.806 packaged in AAV-DJ with a CBA promoter, isefficacious in vivo in down-modulating SOD1 protein levels. In addition,these results demonstrate that a total intrastriatal dose as low as3.7×10⁹ vg of VOYmiR-114.806 packaged in AAVDJ with a CBA promoterresulted in significant down-modulation of SOD1 protein levels.

We claim:
 1. An adeno-associated viral (AAV) vector genome comprising anucleic acid sequence positioned between two inverted terminal repeats(ITRs); wherein said nucleic acid sequence encodes a sense strandsequence and an antisense strand sequence of an siRNA duplex; whereinthe antisense strand sequence comprises nucleotides 1-18 of SEQ ID NO.261; and wherein the sense strand sequence comprises nucleotides 1-17 ofSEQ ID NO.
 93. 2. The AAV vector genome of claim 1, wherein the sensestrand sequence and the antisense strand sequence are, independently,between 19-22 nucleotides in length.
 3. The AAV vector genome of claim1, wherein the antisense strand sequence comprises nucleotides 1-19 ofSEQ ID NO.
 261. 4. The AAV vector genome of claim 1, wherein the sensestrand sequence comprises nucleotides 1-18 of SEQ ID NO.
 93. 5. The AAVvector genome of claim 1, wherein the sense strand sequence comprisesnucleotides 1-19 of SEQ ID NO.
 93. 6. The AAV vector genome of claim 1,wherein the antisense strand sequence comprises nucleotides 1-19 of SEQID NO. 261, and wherein the sense strand sequence comprises nucleotides1-18 of SEQ ID NO.
 93. 7. The AAV vector genome of claim 6, wherein thesense strand sequence comprises nucleotides 1-19 of SEQ ID NO.
 93. 8.The AAV vector genome of claim 7, wherein the sense strand sequence andthe antisense strand sequence are, independently, between 19-22nucleotides in length.
 9. The AAV vector genome of claim 1, wherein thesense strand sequence comprises nucleotides 1-18 of SEQ ID NO.
 92. 10.The AAV vector genome of claim 1, wherein the sense strand sequencecomprises nucleotides 1-19 of SEQ ID NO.
 92. 11. The AAV vector genomeof claim 1, wherein the antisense strand sequence comprises nucleotides1-19 of SEQ ID NO. 261, and wherein the sense strand sequence comprisesnucleotides 1-18 of SEQ ID NO.
 92. 12. The AAV vector genome of claim11, wherein the sense strand sequence comprises nucleotides 1-19 of SEQID NO.
 92. 13. The AAV vector genome of claim 12, wherein the sensestrand sequence and the antisense strand sequence are, independently,between 19-22 nucleotides in length.
 14. The AAV vector genome of claim1, wherein at least one of the sense strand sequence and the antisensestrand sequence comprise a 3′ overhang of at least 1 nucleotide.
 15. TheAAV vector genome of claim 14, wherein the 3′ overhang is adeoxyribonucleotide.
 16. An AAV particle comprising the AAV vectorgenome of claim
 1. 17. The AAV particle of claim 16, comprising anAAVrh10 capsid.
 18. A method for inhibiting the expression of SOD1 genein a cell comprising administering to the cell a composition comprisingan AAV particle of claim
 16. 19. The method of claim 18, wherein thecell is a mammalian motor neuron or astrocyte.
 20. A method for treatingamyotrophic lateral sclerosis (ALS) in a subject, the method comprisingadministering to the subject a therapeutically effective amount of acomposition comprising an AAV particle of claim
 16. 21. The method ofclaim 20, wherein the expression of SOD1 mRNA in a target cell isinhibited or suppressed by about 50% to about 93%.
 22. The method ofclaim 20, wherein the administration of the composition comprisesintraparenchymal spinal administration.
 23. An siRNA duplex comprising asense strand sequence and an antisense strand sequence; wherein theantisense strand sequence comprises nucleotides 1-18 of SEQ ID NO. 261;and wherein the sense strand sequence comprises nucleotides 1-17 of SEQID NO.
 93. 24. The siRNA duplex of claim 23, wherein the antisensestrand sequence comprises nucleotides 1-19 of SEQ ID NO.
 261. 25. ThesiRNA duplex of claim 23, wherein the sense strand sequence comprisesnucleotides 1-19 of SEQ ID NO.
 93. 26. The siRNA duplex of claim 23,wherein the antisense strand sequence comprises nucleotides 1-19 of SEQID NO. 261, and wherein the sense strand sequence comprises nucleotides1-19 of SEQ ID NO.
 93. 27. The siRNA duplex of claim 26, wherein thesense strand sequence and the antisense strand sequence are,independently, between 19-22 nucleotides in length.
 28. The siRNA duplexof claim 23, wherein the sense strand sequence comprises nucleotides1-19 of SEQ ID NO.
 92. 29. The siRNA duplex of claim 23, wherein theantisense strand sequence comprises nucleotides 1-19 of SEQ ID NO. 261,and wherein the sense strand sequence comprises nucleotides 1-19 of SEQID NO.
 92. 30. The siRNA duplex of claim 29, wherein the sense strandsequence and the antisense strand sequence are, independently, between19-22 nucleotides in length.